Moraxella catarrhalis BASB034 polypeptides and used thereof

ABSTRACT

Provided are  Moraxella catarrhalis  BASB034 polypeptides and immunogenic fragments of BASB034 polypeptides. Preferably, the immunogenic fragments have at least 15 amino acids that match an aligned contiguous segment of SEQ ID NOs:2, 4, 6 or 8. The immunogenic fragments, when administered to a subject in a suitable composition (which can include an adjuvant, or a suitable carrier coupled to the fragment) raise an immune response that recognizes a polypeptide having the sequence of SEQ ID NOs:2, 4, 6 or 8. The invention further provides immunogenic compositions containing BASB034 polypeptides and immunogenic fragments thereof, and a pharmaceutically acceptable carrier. Also provided are fusion proteins that contain BASB034 polypeptides and immunogenic fragments thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional application of U.S. patentapplication Ser. No. 09/787,083, filed May 14, 2001, which is theNational Stage Application of International Application No.PCT/EP99/06781, filed Sep. 14, 1999 which was published under PCTarticle 21(2) in English, which claims the benefit of priority of GreatBritain Patent Application Serial No. 9820002.5, filed Sep. 14, 1998.The disclosure of these applications are hereby incorporated byreference as if fully set forth herein.

FIELD OF THE INVENTION

[0002] This invention relates to polynucleotides, (herein referred to as“BASB034 polynucleotide(s)”), polypeptides encoded by them (referred toherein as “BASB034” or “BASB034 polypeptide(s)”), recombinant materialsand methods for their production. In another aspect, the inventionrelates to methods for using such polypeptides and polynucleotides,including vaccines against bacterial infections. In a further aspect,the invention relates to diagnostic assays for detecting infection ofcertain pathogens.

BACKGROUND OF THE INVENTION

[0003]Moraxella catarrhalis (also named Branhamella catarrhalis) is aGram negative bacteria frequently isolated from the human upperrespiratory tract. It is responsible for several pathologies the mainones being otitis media in infants and children, and pneumonia inelderlies. It is also responsible of sinusitis, nosocomial infectionsand less frequently of invasive diseases.

[0004] Otitis media is an important childhood disease both by the numberof cases and its potential sequelae. More than 3.5 millions cases arerecorded every year in the United States, and it is estimated that 80%of the children have experienced at least one episode of otitis beforereaching the age of 3 (Klein, J O (1994) Clin. Inf. Dis 19:823). Leftuntreated, or becoming chronic, this disease may lead to hearing lossesthat could be temporary (in the case of fluid accumulation in the middleear) or permanent (if the auditive nerve is damaged). In infants, suchhearing losses may be responsible for a delayed speech learning.

[0005] Three bacterial species are primarily isolated from the middleear of children with otitis media: Streptococcus pneumoniae, nontypeable Haemophilus influenza (NTHi) and M. catarrhalis. They arepresent in 60 to 90% of the cases. A review of recent studies shows thatS. pneumoniae and NTHi represent both about 30%, and M. catarrhalisabout 15% of the otitis media cases (Murphy, T F (1996) Microbiol. Rev.60:267). Other bacteria could be isolated from the middle ear (H.influenza type B, S. pyogenes etc) but at a much lower frequency (2% ofthe cases or less).

[0006] Epidemiological data indicate that, for the pathogens found inthe middle ear, the colonization of the upper respiratory tract is anabsolute prerequisite for the development of an otitis; other arehowever also required to lead to the disease (Dickinson, D P et al.(1988) J. Infect. Dis. 158:205, Faden, H L et al. (1991) Ann. Otorhinol.Laryngol. 100:612). These are important to trigger the migration of thebacteria into the middle ear via the Eustachian tubes, followed by theinitiation of an inflammatory process. These factors are unknown todate. It has been postulated that a transient anomaly of the immunesystem following a viral infection, for example, could cause aninability to control the colonization of the respiratory tract (Faden, HL et al (1994) J. Infect. Dis. 169:1312). An alternative explanation isthat the exposure to environmental factors allow a more importantcolonization of some children, who subsequently become susceptible tothe development of otitis media because of the sustained presence ofmiddle ear pathogens (Murphy, T F (1996) Microbiol. Rev. 60:267).

[0007] The immune response to M. catarrhalis is poorly characterized.The analysis of strains isolated sequentially from the nasopharynx ofbabies followed from 0 to 2 years of age, indicates that they get andeliminate frequently new strains. This indicates that an efficaciousimmune response against this bacteria is mounted by the colonizedchildren (Faden, H L et al (1994) J. Infect. Dis. 169:1312).

[0008] In most adults tested, bactericidal antibodies have beenidentified (Chapman, A J et al. (1985) J. Infect. Dis. 151:878). Strainsof M. catarrhalis present variations in their capacity to resist serumbactericidal activity: in general, isolates from diseased individualsare more resistant than those who are simply colonized (Hol, C et al.(1993) Lancet 341:1281, Jordan, K L et al. (1990) Am. J. Med. 88 (suppl.5A):28S). Serum resistance could therfore be considered as a virulencefactor of the bacteria. An opsonizing activity has been observed in thesera of children recovering from otitis media.

[0009] The antigens targetted by these different immune responses inhumans have not been identified, with the exception of OMP B 1, a 84 kDaprotein which expression is regulated by iron, and that is recognized bythe sera of patients with pneumonia (Sethi, S, et al. (1995) Infect.Immun. 63:1516), and of UspA1 and UspA2 (Chen D. et al.(1999), Infect.Immun. 67:1310).

[0010] A few other membrane proteins present on the surface of M.catarrhalis have been characterized using biochemical method, or fortheir potential implication in the induction of a protective immunity(for review, see Murphy, T F (1996) Microbiol. Rev. 60:267). In a mousepneumonia model, the presence of antibodies raised against some of them(UspA, CopB) favors a faster clearance of the pulmonary infection.Another polypeptide (OMP CD) is highly conserved among M. catarrhalisstrains, and presents homologies with a porin of Pseudomonas aeruginosa,which has been demonstrated efficacious against this bacterium in animalmodels.

[0011] The frequency of Moraxella catarrhalis infections has risendramatically in the past few decades. This has been attributed to theemergence of multiply antibiotic resistant strains and an increasingpopulation of people with weakened immune systems. It is no longeruncommon to isolate Moraxella catarrhalis strains that are resistant tosome or all of the standard antibiotics. This phenomenon has created anunmet medical need and demand for new anti-microbial agents, vaccines,drug screening methods, and diagnostic tests for this organism.

SUMMARY OF THE INVENTION

[0012] The present invention relates to BASB034, in particular BASB034polypeptides and BASB034 polynucleotides, recombinant materials andmethods for their production. In another aspect, the invention relatesto methods for using such polypeptides and polynucleotides, includingprevention and treatment of microbial diseases, amongst others. In afurther aspect, the invention relates to diagnostic assays for detectingdiseases associated with microbial infections and conditions associatedwith such infections, such as assays for detecting expression oractivity of BASB034 polynucleotides or polypeptides.

[0013] Various changes and modifications within the spirit and scope ofthe disclosed invention will become readily apparent to those skilled inthe art from reading the following descriptions and from reading theother parts of the present disclosure.

DESCRIPTION OF THE INVENTION

[0014] The invention relates to BASB034 polypeptides and polynucleotidesas described in greater detail below. In particular, the inventionrelates to polypeptides and polynucleotides of BASB034 of Moraxellacatarrhalis, which is related by amino acid sequence homology toKlebsiella pneumoniae outer membrane phospholipase A protein. Theinvention relates especially to BASB034 having the nucleotide and aminoacid sequences set out in SEQ ID NO:1, 3, 5 or 7 and SEQ ID NO:2, 4, 6or 8 respectively. It is understood that sequences recited in theSequence Listing below as “DNA” represent an exemplification of oneembodiment of the invention, since those of ordinary skill willrecognize that such sequences can be usefully employed inpolynucleotides in general, including ribopolynucleotides.

[0015] Polypeptides

[0016] In one aspect of the invention there are provided polypeptides ofMoraxella catarrhalis referred to herein as “BASB034” and “BASB034polypeptides” as well as biologically, diagnostically, prophylactically,clinically or therapeutically useful variants thereof, and compositionscomprising the same.

[0017] The present invention further provides for:

[0018] (a) an isolated polypeptide which comprises an amino acidsequence which has at least 85% identity, preferably at least 90%identity, more preferably at least 95% identity, most preferably atleast 97-99% or exact identity, to that of SEQ ID NO:2, 4, 6 or 8;

[0019] (b) a polypeptide encoded by an isolated polynucleotidecomprising a polynucleotide sequence which has at least 85% identity,preferably at least 90% identity, more preferably at least 95% identity,even more preferably at least 97-99% or exact identity to SEQ ID NO:1,3, 5 or 7 over the entire length of SEQ ID NO:1, 3, 5 or 7 respectively;or

[0020] (c) a polypeptide encoded by an isolated polynucleotidecomprising a polynucleotide sequence encoding a polypeptide which has atleast 85% identity, preferably at least 90% identity, more preferably atleast 95% identity, even more preferably at least 97-99% or exactidentity, to the amino acid sequence of SEQ ID NO:2, 4, 6 or 8.

[0021] The BASB034 polypeptides provided in SEQ ID NO:2, 4, 6 or 8 arethe BASB034 polypeptides from Moraxella catarrhalis strains Mc2931 (ATCC43617), Mc2908, Mc2913 and Mc2969.

[0022] The invention also provides an immunogenic fragment of a BASB034polypeptide, that is, a contiguous portion of the BASB034 polypeptidewhich has the same or substantially the same immunogenic activity as thepolypeptide comprising the amino acid sequence of SEQ ID NO:2, 4, 6 or8; That is to say, the fragment (if necessary when coupled to a carrier)is capable of raising an immune response which recognises the BASB034polypeptide. Such an immunogenic fragment may include, for example, theBASB034 polypeptide lacking an N-terminal leader sequence, and/or atransmembrane domain and/or a C-terminal anchor domain. In a preferredaspect the immunogenic fragment of BASB034 according to the inventioncomprises substantially all of the extracellular domain of a polypeptidewhich has at least 85% identity, preferably at least 90% identity, morepreferably at least 95% identity, most preferably at least 97-99%identity, to that of SEQ ID NO:2, 4, 6 or 8 over the entire length ofSEQ ID NO:2

[0023] A fragment is a polypeptide having an amino acid sequence that isentirely the same as part but not all of any amino acid sequence of anypolypeptide of the invention. As with BASB034 polypeptides, fragmentsmay be “free-standing,” or comprised within a larger polypeptide ofwhich they form a part or region, most preferably as a single continuousregion in a single larger polypeptide.

[0024] Preferred fragments include, for example, truncation polypeptideshaving a portion of an amino acid sequence of SEQ ID NO:2, 4, 6 or 8 orof variants thereof, such as a continuous series of residues thatincludes an amino- and/or carboxyl-terminal amino acid sequence.Degradation forms of the polypeptides of the invention produced by or ina host cell, are also preferred. Further preferred are fragmentscharacterized by structural or functional attributes such as fragmentsthat comprise alpha-helix and alpha-helix forming regions, beta-sheetand beta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions.

[0025] Further preferred fragments include an isolated polypeptidecomprising an amino acid sequence having at least 15, 20, 30, 40, 50 or100 contiguous amino acids from the amino acid sequence of SEQ ID NO:2,4, 6 or 8, or an isolated polypeptide comprising an amino acid sequencehaving at least 15, 20, 30, 40, 50 or 100 contiguous amino acidstruncated or deleted from the amino acid sequence of SEQ ID NO:2, 4, 6or 8.

[0026] Fragments of the polypeptides of the invention may be employedfor producing the corresponding full-length polypeptide by peptidesynthesis; therefore, these fragments may be employed as intermediatesfor producing the full-length polypeptides of the invention.

[0027] Particularly preferred are variants in which several, 5-10, 1-5,1-3, 1-2 or 1 amino acids are substituted, deleted, or added in anycombination.

[0028] The polypeptides, or immunogenic fragments, of the invention maybe in the form of the “mature” protein or may be a part of a largerprotein such as a precursor or a fusion protein. It is oftenadvantageous to include an additional amino acid sequence which containssecretory or leader sequences, pro-sequences, sequences which aid inpurification such as multiple histidine residues, or an additionalsequence for stability during recombinant production. Furthermore,addition of exogenous polypeptide or lipid tail or polynucleotidesequences to increase the immunogenic potential of the final molecule isalso considered.

[0029] In one aspect, the invention relates to genetically engineeredsoluble fusion proteins comprising a polypeptide of the presentinvention, or a fragment thereof, and various portions of the constantregions of heavy or light chains of immunoglobulins of varioussubclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is theconstant part of the heavy chain of human IgG, particularly IgG1, wherefusion takes place at the hinge region. In a particular embodiment, theFc part can be removed simply by incorporation of a cleavage sequencewhich can be cleaved with blood clotting factor Xa.

[0030] Furthermore, this invention relates to processes for thepreparation of these fusion proteins by genetic engineering, and to theuse thereof for drug screening, diagnosis and therapy. A further aspectof the invention also relates to polynucleotides encoding such fusionproteins. Examples of fusion protein technology can be found inInternational Patent Application Nos. WO94/29458 and WO94/22914.

[0031] The proteins may be chemically conjugated, or expressed asrecombinant fusion proteins allowing increased levels to be produced inan expression system as compared to non-fused protein. The fusionpartner may assist in providing T helper epitopes (immunological fusionpartner), preferably T helper epitopes recognised by humans, or assistin expressing the protein (expression enhancer) at higher yields thanthe native recombinant protein. Preferably the fusion partner will beboth an immunological fusion partner and expression enhancing partner.

[0032] Fusion partners include protein D from Haemophilus influenzae andthe non-structural protein from influenzae virus, NS1 (hemagglutinin).Another fusion partner is the protein known as LytA. Preferably the Cterminal portion of the molecule is used. Lyta is derived fromStreptococcus pneumoniae which synthesize an N-acetyl-L-alanine amidase,amidase LytA, (coded by the lytA gene {Gene, 43 (1986) page 265-272}) anautolysin that specifically degrades certain bonds in the peptidoglycanbackbone. The C-terminal domain of the LytA protein is responsible forthe affinity to the choline or to some choline analogues such as DEAE.This property has been exploited for the development of E. coli C-LytAexpressing plasmids useful for expression of fusion proteins.Purification of hybrid proteins containing the C-LytA fragment at itsamino terminus has been described {Biotechnology: 10, (1992) page795-798}. It is possible to use the repeat portion of the LytA moleculefound in the C terminal end starting at residue 178, for exampleresidues 188-305.

[0033] The present invention also includes variants of theaforementioned polypeptides, that is polypeptides that vary from thereferents by conservative amino acid substitutions, whereby a residue issubstituted by another with like characteristics. Typical suchsubstitutions are among Ala, Val, Leu and Ile; among Ser and Thr; amongthe acidic residues Asp and Glu; among Asn and Gln; and among the basicresidues Lys and Arg; or aromatic residues Phe and Tyr.

[0034] Polypeptides of the present invention can be prepared in anysuitable manner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

[0035] It is most preferred that a polypeptide of the invention isderived from Moraxella catarrhalis, however, it may preferably beobtained from other organisms of the same taxonomic genus. A polypeptideof the invention may also be obtained, for example, from organisms ofthe same taxonomic family or order.

[0036] Polynucleotides

[0037] It is an object of the invention to provide polynucleotides thatencode BASB034 polypeptides, particularly polynucleotides that encodethe polypeptide herein designated BASB034.

[0038] In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding BASB034 polypeptidescomprising a sequence set out in SEQ ID NO:1, 3, 5 or 7 which includes afull length gene, or a variant thereof.

[0039] The BASB034 polynucleotides provided in SEQ ID NO:1, 3, 5 or 7are the BASB034 polynucleotides from Moraxella catarrhalis strainsMc2931 (ATCC 43617), Mc2908, Mc2913 and Mc2969.

[0040] As a further aspect of the invention there are provided isolatednucleic acid molecules encoding and/or expressing BASB034 polypeptidesand polynucleotides, particularly Moraxella catarrhalis BASB034polypeptides and polynucleotides, including, for example, unprocessedRNAs, ribozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs. Furtherembodiments of the invention include biologically, diagnostically,prophylactically, clinically or therapeutically useful polynucleotidesand polypeptides, and variants thereof, and compositions comprising thesame.

[0041] Another aspect of the invention relates to isolatedpolynucleotides, including at least one full length gene, that encodes aBASB034 polypeptide having a deduced amino acid sequence of SEQ ID NO:2,4, 6 or 8 and polynucleotides closely related thereto and variantsthereof.

[0042] In another particularly preferred embodiment of the inventionthere is a BASB034 polypeptide from Moraxella catarrhalis comprising orconsisting of an amino acid sequence of SEQ ID NO:2, 4, 6 or 8 or avariant thereof.

[0043] Using the information provided herein, such as a polynucleotidesequence set out in SEQ ID NO:1, 3, 5 or 7, a polynucleotide of theinvention encoding BASB034 polypeptide may be obtained using standardcloning and screening methods, such as those for cloning and sequencingchromosomal DNA fragments from bacteria using Moraxella catarrhalisCatlin cells as starting material, followed by obtaining a full lengthclone. For example, to obtain a polynucleotide sequence of theinvention, such as a polynucleotide sequence given in SEQ ID NO:1, 3, 5or 7, typically a library of clones of chromosomal DNA of Moraxellacatarrhalis Catlin in E. coli or some other suitable host is probed witha radiolabeled oligonucleotide, preferably a 17-mer or longer, derivedfrom a partial sequence. Clones carrying DNA identical to that of theprobe can then be distinguished using stringent hybridizationconditions. By sequencing the individual clones thus identified byhybridization with sequencing primers designed from the originalpolypeptide or polynucleotide sequence it is then possible to extend thepolynucleotide sequence in both directions to determine a full lengthgene sequence. Conveniently, such sequencing is performed, for example,using denatured double stranded DNA prepared from a plasmid clone.Suitable techniques are described by Maniatis, T., Fritsch, E. F. andSambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). (see inparticular Screening By Hybridization 1.90 and Sequencing DenaturedDouble-Stranded DNA Templates 13.70). Direct genomic DNA sequencing mayalso be performed to obtain a full length gene sequence. Illustrative ofthe invention, each polynucleotide set out in SEQ ID NO:1, 3, 5 or 7 wasdiscovered in a DNA library derived from Moraxella catarrhalis.

[0044] Moreover, each DNA sequence set out in SEQ ID NO:1, 3, 5 or 7contains an open reading frame encoding a protein having about thenumber of amino acid residues set forth in SEQ ID NO:2, 4, 6 or 8 with adeduced molecular weight that can be calculated using amino acid residuemolecular weight values well known to those skilled in the art.

[0045] The polynucleotide of SEQ ID NO:1, between the start codon atnucleotide number 1 and the stop codon which begins at nucleotide number1327 of SEQ ID NO:1, encodes the polypeptide of SEQ ID NO:2.

[0046] The polynucleotide of SEQ ID NO:3, between the start codon atnucleotide number 1 and the stop codon which begins at nucleotide number1327 of SEQ ID NO:3, encodes the polypeptide of SEQ ID NO:4.

[0047] The polynucleotide of SEQ ID NO:5, between the start codon atnucleotide number 1 and the stop codon which begins at nucleotide number1327 of SEQ ID NO:5, encodes the polypeptide of SEQ ID NO:6.

[0048] The polynucleotide of SEQ ID NO:7, between the start codon atnucleotide number 1 and the stop codon which begins at nucleotide number1327 of SEQ ID NO:7, encodes the polypeptide of SEQ ID NO:8.

[0049] In a further aspect, the present invention provides for anisolated polynucleotide comprising or consisting of:

[0050] (a) a polynucleotide sequence which has at least 85% identity,preferably at least 90% identity, more preferably at least 95% identity,even more preferably at least 97-99% or exact identity to SEQ ID NO:1,3, 5 or 7 over the entire length of SEQ ID NO:1, 3, 5 or 7 respectively;or

[0051] (b) a polynucleotide sequence encoding a polypeptide which has atleast 85% identity, preferably at least 90% identity, more preferably atleast 95% identity, even more preferably at least 97-99% or 100% exact,to the amino acid sequence of SEQ ID NO:2, 4, 6 or 8, over the entirelength of SEQ ID NO:2, 4, 6 or 8 respectively.

[0052] A polynucleotide encoding a polypeptide of the present invention,including homologs and orthologs from species other than Moraxellacatarrhalis, may be obtained by a process which comprises the steps ofscreening an appropriate library under stringent hybridizationconditions (for example, using a temperature in the range of 45-65° C.and an SDS concentration from 0.1-1%) with a labeled or detectable probeconsisting of or comprising the sequence of SEQ ID NO:1, 3, 5 or 7 or afragment thereof; and isolating a full-length gene and/or genomic clonescontaining said polynucleotide sequence.

[0053] The invention provides a polynucleotide sequence identical overits entire length to a coding sequence (open reading frame) in SEQ IDNO:1, 3, 5 or 7. Also provided by the invention is a coding sequence fora mature polypeptide or a fragment thereof, by itself as well as acoding sequence for a mature polypeptide or a fragment in reading framewith another coding sequence, such as a sequence encoding a leader orsecretory sequence, a pre-, or pro- or prepro-protein sequence. Thepolynucleotide of the invention may also contain at least one non-codingsequence, including for example, but not limited to at least onenon-coding 5′ and 3′ sequence, such as the transcribed butnon-translated sequences, termination signals (such as rho-dependent andrho-independent termination signals), ribosome binding sites, Kozaksequences, sequences that stabilize mRNA, introns, and polyadenylationsignals. The polynucleotide sequence may also comprise additional codingsequence encoding additional amino acids. For example, a marker sequencethat facilitates purification of the fused polypeptide can be encoded.In certain embodiments of the invention, the marker sequence is ahexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) anddescribed in Gentz et al., Proc. Natl. Acad. Sci., USA 86: 821-824(1989), or an HA peptide tag (Wilson et al., Cell 37: 767 (1984), bothof which may be useful in purifying polypeptide sequence fused to them.Polynucleotides of the invention also include, but are not limited to,polynucleotides comprising a structural gene and its naturallyassociated sequences that control gene expression.

[0054] The nucleotide sequence encoding BASB034 polypeptide of SEQ IDNO:2, 4, 6 or 8 may be identical to the polypeptide encoding sequencecontained in nucleotides 1 to 1326 of SEQ ID NO:1, 3, 5 or 7respectively. Alternatively it may be a sequence, which as a result ofthe redundancy (degeneracy) of the genetic code, also encodes thepolypeptide of SEQ ID NO:2, 4, 6 or 8.

[0055] The term “polynucleotide encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Moraxella catarrhalis BASB034having an amino acid sequence set out in SEQ ID NO:2, 4, 6 or 8. Theterm also encompasses polynucleotides that include a single continuousregion or discontinuous regions encoding the polypeptide (for example,polynucleotides interrupted by integrated phage, an integrated insertionsequence, an integrated vector sequence, an integrated transposonsequence, or due to RNA editing or genomic DNA reorganization) togetherwith additional regions, that also may contain coding and/or non-codingsequences.

[0056] The invention further relates to variants of the polynucleotidesdescribed herein that encode variants of a polypeptide having a deducedamino acid sequence of SEQ ID NO:2, 4, 6 or 8. Fragments ofpolynucleotides of the invention may be used, for example, to synthesizefull-length polynucleotides of the invention.

[0057] Further particularly preferred embodiments are polynucleotidesencoding BASB034 variants, that have the amino acid sequence of BASB034polypeptide of SEQ ID NO:2, 4, 6 or 8 in which several, a few, 5 to 10,1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted,modified, deleted and/or added, in any combination. Especially preferredamong these are silent substitutions, additions and deletions, that donot alter the properties and activities of BASB034 polypeptide.

[0058] Further preferred embodiments of the invention arepolynucleotides that are at least 85% identical over their entire lengthto a polynucleotide encoding BASB034 polypeptide having an amino acidsequence set out in SEQ ID NO:2, 4, 6 or 8, and polynucleotides that arecomplementary to such polynucleotides. Alternatively, most highlypreferred are polynucleotides that comprise a region that is at least90% identical over its entire length to a polynucleotide encodingBASB034 polypeptide and polynucleotides complementary thereto. In thisregard, polynucleotides at least 95% identical over their entire lengthto the same are particularly preferred. Furthermore, those with at least97% are highly preferred among those with at least 95%, and among thesethose with at least 98% and at least 99% are particularly highlypreferred, with at least 99% being the more preferred.

[0059] Preferred embodiments are polynucleotides encoding polypeptidesthat retain substantially the same biological function or activity asthe mature polypeptide encoded by a DNA of SEQ ID NO:1, 3, 5 or 7.

[0060] In accordance with certain preferred embodiments of thisinvention there are provided polynucleotides that hybridize,particularly under stringent conditions, to BASB034 polynucleotidesequences, such as those polynucleotides in SEQ ID NO:1, 3, 5 or 7.

[0061] The invention further relates to polynucleotides that hybridizeto the polynucleotide sequences provided herein. In this regard, theinvention especially relates to polynucleotides that hybridize understringent conditions to the polynucleotides described herein. As hereinused, the terms “stringent conditions” and “stringent hybridizationconditions” mean hybridization occurring only if there is at least 95%and preferably at least 97% identity between the sequences. A specificexample of stringent hybridization conditions is overnight incubation at42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml of denatured,sheared salmon sperm DNA, followed by washing the hybridization supportin 0.1×SSC at about 65° C. Hybridization and wash conditions are wellknown and exemplified in Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989),particularly Chapter 11 therein. Solution hybridization may also be usedwith the polynucleotide sequences provided by the invention.

[0062] The invention also provides a polynucleotide consisting of orcomprising a polynucleotide sequence obtained by screening anappropriate library containing the complete gene for a polynucleotidesequence set forth in SEQ ID NO:1, 3, 5 or 7 under stringenthybridization conditions with a probe having the sequence of saidpolynucleotide sequence set forth in SEQ ID NO:1, 3, 5 or 7 or afragment thereof; and isolating said polynucleotide sequence. Fragmentsuseful for obtaining such a polynucleotide include, for example, probesand primers fully described elsewhere herein.

[0063] As discussed elsewhere herein regarding polynucleotide assays ofthe invention, for instance, the polynucleotides of the invention, maybe used as a hybridization probe for RNA, cDNA and genomic DNA toisolate full-length cDNAs and genomic clones encoding BASB034 and toisolate cDNA and genomic clones of other genes that have a highidentity, particularly high sequence identity, to the BASB034 gene. Suchprobes generally will comprise at least 15 nucleotide residues or basepairs. Preferably, such probes will have at least 30 nucleotide residuesor base pairs and may have at least 50 nucleotide residues or basepairs. Particularly preferred probes will have at least 20 nucleotideresidues or base pairs and will have less than 30 nucleotide residues orbase pairs.

[0064] A coding region of BASB034 gene may be isolated by screeningusing a DNA sequence provided in SEQ ID NO:1, 3, 5 or 7 to synthesize anoligonucleotide probe. A labeled oligonucleotide have a sequencecomplementary to that of a gene of the invention is then used to screena library of cDNA, genomic DNA or mRNA to determine which members of thelibrary the probe hybridizes to.

[0065] There are several methods available and well known to thoseskilled in the art to obtain full-length DNAs, or extend short DNAs, forexample those based on the method of Rapid Amplification of cDNA ends(RACE) (see, for example, Frohman, et al., PNAS USA 85: 8998-9002,1988). Recent modifications of the technique, exemplified by theMarathon™ technology (Clontech Laboratories Inc.) for example, havesignificantly simplified the search for longer cDNAs. In the Marathon™technology, cDNAs have been prepared from mRNA extracted from a chosentissue and an ‘adaptor’ sequence ligated onto each end. Nucleic acidamplification (PCR) is then carried out to amplify the “missing” 5′ endof the DNA using a combination of gene specific and adaptor specificoligonucleotide primers. The PCR reaction is then repeated using“nested” primers, that is, primers designed to anneal within theamplified product (typically an adaptor specific primer that annealsfurther 3′ in the adaptor sequence and a gene specific primer thatanneals further 5′ in the selected gene sequence). The product of thisreaction can then be analyzed by DNA sequencing and a full-length DNAconstructed either by joining the product directly to the existing DNAto give a complete sequence, or carrying out a separate full-length PCRusing the new sequence information for the design of the 5′ primer.

[0066] The polynucleotides and polypeptides of the invention may beemployed, for example, as research reagents and materials for discoveryof treatments of and diagnostics for diseases, particularly humandiseases, as further discussed herein relating to polynucleotide assays.

[0067] The polynucleotides of the invention that are oligonucleotidesderived from a sequence of SEQ ID NOS:1-8 may be used in the processesherein as described, but preferably for PCR, to determine whether or notthe polynucleotides identified herein in whole or in part aretranscribed in bacteria in infected tissue. It is recognized that suchsequences will also have utility in diagnosis of the stage of infectionand type of infection the pathogen has attained.

[0068] The invention also provides polynucleotides that encode apolypeptide that is the mature protein plus additional amino orcarboxyl-terminal amino acids, or amino acids interior to the maturepolypeptide (when the mature form has more than one polypeptide chain,for instance). Such sequences may play a role in processing of a proteinfrom precursor to a mature form, may allow protein transport, maylengthen or shorten protein half-life or may facilitate manipulation ofa protein for assay or production, among other things. As generally isthe case in vivo, the additional amino acids may be processed away fromthe mature protein by cellular enzymes.

[0069] For each and every polynucleotide of the invention there isprovided a polynucleotide complementary to it. It is preferred thatthese complementary polynucleotides are fully complementary to eachpolynucleotide with which they are complementary.

[0070] A precursor protein, having a mature form of the polypeptidefused to one or more prosequences may be an inactive form of thepolypeptide. When prosequences are removed such inactive precursorsgenerally are activated. Some or all of the prosequences may be removedbefore activation. Generally, such precursors are called proproteins.

[0071] In addition to the standard A, G, C, T/U representations fornucleotides, the term “N” may also be used in describing certainpolynucleotides of the invention. “N” means that any of the four DNA orRNA nucleotides may appear at such a designated position in the DNA orRNA sequence, except it is preferred that N is not a nucleic acid thatwhen taken in combination with adjacent nucleotide positions, when readin the correct reading frame, would have the effect of generating apremature termination codon in such reading frame.

[0072] In sum, a polynucleotide of the invention may encode a matureprotein, a mature protein plus a leader sequence (which may be referredto as a preprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

[0073] In accordance with an aspect of the invention, there is providedthe use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization.

[0074] The use of a polynucleotide of the invention in geneticimmunization will preferably employ a suitable delivery method such asdirect injection of plasmid DNA into muscles (Wolff et al., Hum MolGenet (1992) 1: 363, Manthorpe et al., Hum. Gene Ther. (1983) 4: 419),delivery of DNA complexed with specific protein carriers (Wu et al., JBiol Chem. (1989) 264: 16985), coprecipitation of DNA with calciumphosphate (Benvenisty & Reshef, PNAS USA, (1986) 83: 9551),encapsulation of DNA in various forms of liposomes (Kaneda et al.,Science (1989) 243: 375), particle bombardment (Tang et al., Nature(1992) 356:152, Eisenbraun et al., DNA Cell Biol (1993) 12: 791) and invivo infection using cloned retroviral vectors (Seeger et al., PNAS USA(1984) 81: 5849).

[0075] Vectors, Host Cells, Expression Systems

[0076] The invention also relates to vectors that comprise apolynucleotide or polynucleotides of the invention, host cells that aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

[0077] Recombinant polypeptides of the present invention may be preparedby processes well known in those skilled in the art from geneticallyengineered host cells comprising expression systems. Accordingly, in afurther aspect, the present invention relates to expression systems thatcomprise a polynucleotide or polynucleotides of the present invention,to host cells which are genetically engineered with such expressionsystems, and to the production of polypeptides of the invention byrecombinant techniques.

[0078] For recombinant production of the polypeptides of the invention,host cells can be genetically engineered to incorporate expressionsystems or portions thereof or polynucleotides of the invention.Introduction of a polynucleotide into the host cell can be effected bymethods described in many standard laboratory manuals, such as Davis, etal., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al.,MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989), such as, calciumphosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introductionand infection.

[0079] Representative examples of appropriate hosts include bacterialcells, such as cells of streptococci, staphylococci, enterococci, E.coli, streptomyces, cyanobacteria, Bacillus subtilis, Neisseriameningitidis and Moraxella catarrhalis; fungal cells, such as cells of ayeast, Kluveromyces, Saccharomyces, a basidiomycete, Candida albicansand Aspergillus; insect cells such as cells of Drosophila S2 andSpodoptera Sf9; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK,293, CV-1 and Bowes melanoma cells; and plant cells, such as cells of agymnosperm or angiosperm.

[0080] A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal-, episomal- and virus-derived vectors, for example, vectorsderived from bacterial plasmids, from bacteriophage, from transposons,from yeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses, picomaviruses, retroviruses, and alphaviruses and vectorsderived from combinations thereof, such as those derived from plasmidand bacteriophage genetic elements, such as cosmids and phagemids. Theexpression system constructs may contain control regions that regulateas well as engender expression. Generally, any system or vector suitableto maintain, propagate or express polynucleotides and/or to express apolypeptide in a host may be used for expression in this regard. Theappropriate DNA sequence may be inserted into the expression system byany of a variety of well-known and routine techniques, such as, forexample, those set forth in Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, (supra).

[0081] In recombinant expression systems in eukaryotes, for secretion ofa translated protein into the lumen of the endoplasmic reticulum, intothe periplasmic space or into the extracellular environment, appropriatesecretion signals may be incorporated into the expressed polypeptide.These signals may be endogenous to the polypeptide or they may beheterologous signals.

[0082] Polypeptides of the present invention can be recovered andpurified from recombinant cell cultures by well-known methods includingammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxylapatite chromatography and lectin chromatography. Mostpreferably, ion metal affinity chromatography (IMAC) is employed forpurification. Well known techniques for refolding proteins may beemployed to regenerate active conformation when the polypeptide isdenatured during intracellular synthesis, isolation and or purification.

[0083] The expression system may also be a recombinant livemicroorganism, such as a virus or bacterium. The gene of interest can beinserted into the genome of a live recombinant virus or bacterium.Inoculation and in vivo infection with this live vector will lead to invivo expression of the antigen and induction of immune responses.Viruses and bacteria used for this purpose are for instance: poxviruses(e.g; vaccinia, fowlpox, canarypox), alphaviruses (Sindbis virus,Semliki Forest Virus, Venezuelian Equine Encephalitis Virus),adenoviruses, adeno-associated virus, picomaviruses (poliovirus,rhinovirus), herpesviruses (varicella zoster virus, etc), Listeria,Salmonella, Shigella, BCG. These viruses and bacteria can be virulent,or attenuated in various ways in order to obtain live vaccines. Suchlive vaccines also form part of the invention.

[0084] Diagnostic, Prognostic, Serotyping and Mutation Assays

[0085] This invention is also related to the use of BASB034polynucleotides and polypeptides of the invention for use as diagnosticreagents. Detection of BASB034 polynucleotides and/or polypeptides in aeukaryote, particularly a mammal, and especially a human, will provide adiagnostic method for diagnosis of disease, staging of disease orresponse of an infectious organism to drugs. Eukaryotes, particularlymammals, and especially humans, particularly those infected or suspectedto be infected with an organism comprising the BASB034 gene or protein,may be detected at the nucleic acid or amino acid level by a variety ofwell known techniques as well as by methods provided herein.

[0086] Polypeptides and polynucleotides for prognosis, diagnosis orother analysis may be obtained from a putatively infected and/orinfected individual's bodily materials. Polynucleotides from any ofthese sources, particularly DNA or RNA, may be used directly fordetection or may be amplified enzymatically by using PCR or any otheramplification technique prior to analysis. RNA, particularly mRNA, cDNAand genomic DNA may also be used in the same ways. Using amplification,characterization of the species and strain of infectious or residentorganism present in an individual, may be made by an analysis of thegenotype of a selected polynucleotide of the organism. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to a genotype of a reference sequence selected from arelated organism, preferably a different species of the same genus or adifferent strain of the same species. Point mutations can be identifiedby hybridizing amplified DNA to labeled BASB034 polynucleotidesequences. Perfectly or significantly matched sequences can bedistinguished from imperfectly or more significantly mismatched duplexesby DNase or RNase digestion, for DNA or RNA respectively, or bydetecting differences in melting temperatures or renaturation kinetics.Polynucleotide sequence differences may also be detected by alterationsin the electrophoretic mobility of polynucleotide fragments in gels ascompared to a reference sequence. This may be carried out with orwithout denaturing agents. Polynucleotide differences may also bedetected by direct DNA or RNA sequencing. See, for example, Myers etal., Science, 230: 1242 (1985). Sequence changes at specific locationsalso may be revealed by nuclease protection assays, such as RNase, V1and S1 protection assay or a chemical cleavage method. See, for example,Cotton et al., Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985).

[0087] In another embodiment, an array of oligonucleotides probescomprising BASB034 nucleotide sequence or fragments thereof can beconstructed to conduct efficient screening of, for example, geneticmutations, serotype, taxonomic classification or identification. Arraytechnology methods are well known and have general applicability and canbe used to address a variety of questions in molecular geneticsincluding gene expression, genetic linkage, and genetic variability(see, for example, Chee et al., Science, 274: 610 (1996)).

[0088] Thus in another aspect, the present invention relates to adiagnostic kit which comprises:

[0089] (a) a polynucleotide of the present invention, preferably thenucleotide sequence of SEQ ID NO:1, 3, 5 or 7, or a fragment thereof;

[0090] (b) a nucleotide sequence complementary to that of (a);

[0091] (c) a polypeptide of the present invention, preferably thepolypeptide of SEQ ID NO:2, 4, 6 or 8 or a fragment thereof; or

[0092] (d) an antibody to a polypeptide of the present invention,preferably to the polypeptide of SEQ ID NO:2, 4, 6 or 8.

[0093] It will be appreciated that in any such kit, (a), (b), (c) or (d)may comprise a substantial component. Such a kit will be of use indiagnosing a disease or susceptibility to a Disease, among others.

[0094] This invention also relates to the use of polynucleotides of thepresent invention as diagnostic reagents. Detection of a mutated form ofa polynucleotide of the invention, preferable, SEQ ID NO:1, 3, 5 or 7,which is associated with a disease or pathogenicity will provide adiagnostic tool that can add to, or define, a diagnosis of a disease, aprognosis of a course of disease, a determination of a stage of disease,or a susceptibility to a disease, which results from under-expression,over-expression or altered expression of the polynucleotide. Organisms,particularly infectious organisms, carrying mutations in suchpolynucleotide may be detected at the polynucleotide level by a varietyof techniques, such as those described elsewhere herein.

[0095] Cells from an organism carrying mutations or polymorphisms(allelic variations) in a polynucleotide and/or polypeptide of theinvention may also be detected at the polynucleotide or polypeptidelevel by a variety of techniques, to allow for serotyping, for example.For example, RT-PCR can be used to detect mutations in the RNA. It isparticularly preferred to use RT-PCR in conjunction with automateddetection systems, such as, for example, GeneScan. RNA, cDNA or genomicDNA may also be used for the same purpose, PCR. As an example, PCRprimers complementary to a polynucleotide encoding BASB034 polypeptidecan be used to identify and analyze mutations.

[0096] The invention further provides primers with 1, 2, 3 or 4nucleotides removed from the 5′ and/or the 3′ end. These primers may beused for, among other things, amplifying BASB034 DNA and/or RNA isolatedfrom a sample derived from an individual, such as a bodily material. Theprimers may be used to amplify a polynucleotide isolated from aninfected individual, such that the polynucleotide may then be subject tovarious techniques for elucidation of the polynucleotide sequence. Inthis way, mutations in the polynucleotide sequence may be detected andused to diagnose and/or prognose the infection or its stage or course,or to serotype and/or classify the infectious agent.

[0097] The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections caused byMoraxella catarrhalis, comprising determining from a sample derived froman individual, such as a bodily material, an increased level ofexpression of polynucleotide having a sequence of SEQ ID NO:1, 3, 5 or7. Increased or decreased expression of a BASB034 polynucleotide can bemeasured using any on of the methods well known in the art for thequantitation of polynucleotides, such as, for example, amplification,PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and otherhybridization methods.

[0098] In addition, a diagnostic assay in accordance with the inventionfor detecting over-expression of BASB034 polypeptide compared to normalcontrol tissue samples may be used to detect the presence of aninfection, for example. Assay techniques that can be used to determinelevels of a BASB034 polypeptide, in a sample derived from a host, suchas a bodily material, are well-known to those of skill in the art. Suchassay methods include radioimmunoassays, competitive-binding assays,Western Blot analysis, antibody sandwich assays, antibody detection andELISA assays.

[0099] The polynucleotides of the invention may be used as components ofpolynucleotide arrays, preferably high density arrays or grids. Thesehigh density arrays are particularly useful for diagnostic andprognostic purposes. For example, a set of spots each comprising adifferent gene, and further comprising a polynucleotide orpolynucleotides of the invention, may be used for probing, such as usinghybridization or nucleic acid amplification, using probes obtained orderived from bodily sample to determine the presence of a particularpolynucleotide sequence or related sequence in an individual. Such apresence may indicate the presence of a pathogen, particularly Moraxellacatarrhalis, and may be useful in diagnosing and/or prognosing diseaseor a course of disease. A grid comprising a number of variants of thepolynucleotide sequence of SEQ ID NO:1, 3, 5 or 7 are preferred. Alsopreferred is a grid comprising a number of variants of a polynucleotidesequence encoding the polypeptide of SEQ ID NO:2, 4, 6, or 8.

[0100] Antibodies

[0101] The polypeptides and polynucleotides of the invention or variantsthereof, or cells expressing the same can be used as immunogens toproduce antibodies immunospecific for such polypeptides orpolynucleotides respectively.

[0102] In certain preferred embodiments of the invention there areprovided antibodies against BASB034 polypeptides or polynucleotides.

[0103] Antibodies generated against the polypeptides or polynucleotidesof the invention can be obtained by administering the polypeptidesand/or polynucleotides of the invention, or epitope-bearing fragments ofeither or both, analogues of either or both, or cells expressing eitheror both, to an animal, preferably a nonhuman, using routine protocols.For preparation of monoclonal antibodies, any technique known in the artthat provides antibodies produced by continuous cell line cultures canbe used. Examples include various techniques, such as those in Kohler,G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor et al.,Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

[0104] Techniques for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies topolypeptides or polynucleotides of this invention. Also, transgenicmice, or other organisms or animals, such as other mammals, may be usedto express humanized antibodies immunospecific to the polypeptides orpolynucleotides of the invention.

[0105] Alternatively, phage display technology may be utilized to selectantibody genes with binding activities towards a polypeptide of theinvention either from repertoires of PCR amplified v-genes oflymphocytes from humans screened for possessing anti-BASB034 or fromnaive libraries (McCafferty, et al., (1990), Nature 348, 552-554; Marks,et al., (1992) Biotechnology 10, 779-783). The affinity of theseantibodies can also be improved by, for example, chain shuffling(Clackson et al., (1991) Nature 352: 628).

[0106] The above-described antibodies may be employed to isolate or toidentify clones expressing the polypeptides or polynucleotides of theinvention to purify the polypeptides or polynucleotides by, for example,affinity chromatography.

[0107] Thus, among others, antibodies against BASB034-polypeptide orBASB034-polynucleotide may be employed to treat infections, particularlybacterial infections.

[0108] Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants form a particular aspect of thisinvention.

[0109] Preferably, the antibody or variant thereof is modified to makeit less immunogenic in the individual. For example, if the individual ishuman the antibody may most preferably be “humanized,” where thecomplimentarity determining region or regions of the hybridoma-derivedantibody has been transplanted into a human monoclonal antibody, forexample as described in Jones et al. (1986), Nature 321, 522-525 orTempest et al., (1991) Biotechnology 9, 266-273.

[0110] Antagonists and Agonists—Assays and Molecules

[0111] Polypeptides and polynucleotides of the invention may also beused to assess the binding of small molecule substrates and ligands in,for example, cells, cell-free preparations, chemical libraries, andnatural product mixtures. These substrates and ligands may be naturalsubstrates and ligands or may be structural or functional mimetics. See,e.g., Coligan et al, Current Protocols in Immunology 1(2): Chapter 5(1991).

[0112] The screening methods may simply measure the binding of acandidate compound to the polypeptide or polynucleotide, or to cells ormembranes bearing the polypeptide or polynucleotide, or a fusion proteinof the polypeptide by means of a label directly or indirectly associatedwith the candidate compound. Alternatively, the screening method mayinvolve competition with a labeled competitor. Further, these screeningmethods may test whether the candidate compound results in a signalgenerated by activation or inhibition of the polypeptide orpolynucleotide, using detection systems appropriate to the cellscomprising the polypeptide or polynucleotide. Inhibitors of activationare generally assayed in the presence of a known agonist and the effecton activation by the agonist by the presence of the candidate compoundis observed. Constitutively active polypeptide and/or constitutivelyexpressed polypeptides and polynucleotides may be employed in screeningmethods for inverse agonists or inhibitors, in the absence of an agonistor inhibitor, by testing whether the candidate compound results ininhibition of activation of the polypeptide or polynucleotide, as thecase may be. Further, the screening methods may simply comprise thesteps of mixing a candidate compound with a solution containing apolypeptide or polynucleotide of the present invention, to form amixture, measuring BASB034 polypeptide and/or polynucleotide activity inthe mixture, and comparing the BASB034 polypeptide and/or polynucleotideactivity of the mixture to a standard. Fusion proteins, such as thosemade from Fe portion and BASB034 polypeptide, as hereinbefore described,can also be used for high-throughput screening assays to identifyantagonists of the polypeptide of the present invention, as well as ofphylogenetically and and/or functionally related polypeptides (see D.Bennett et al., J Mol Recognition, 8:52-58 (1995); and K. Johanson etal., J Biol Chem, 270(16):9459-9471 (1995)).

[0113] The polynucleotides, polypeptides and antibodies that bind toand/or interact with a polypeptide of the present invention may also beused to configure screening methods for detecting the effect of addedcompounds on the production of mRNA and/or polypeptide in cells. Forexample, an ELISA assay may be constructed for measuring secreted orcell associated levels of polypeptide using monoclonal and polyclonalantibodies by standard methods known in the art. This can be used todiscover agents which may inhibit or enhance the production ofpolypeptide (also called antagonist or agonist, respectively) fromsuitably manipulated cells or tissues.

[0114] The invention also provides a method of screening compounds toidentify those which enhance (agonist) or block (antagonist) the actionof BASB034 polypeptides or polynucleotides, particularly those compoundsthat are bacteriostatic and/or bactericidal. The method of screening mayinvolve high-throughput techniques. For example, to screen for agonistsor antagonists, a synthetic reaction mix, a cellular compartment, suchas a membrane, cell envelope or cell wall, or a preparation of anythereof, comprising BASB034 polypeptide and a labeled substrate orligand of such polypeptide is incubated in the absence or the presenceof a candidate molecule that may be a BASB034 agonist or antagonist. Theability of the candidate molecule to agonize or antagonize the BASB034polypeptide is reflected in decreased binding of the labeled ligand ordecreased production of product from such substrate. Molecules that bindgratuitously, i.e., without inducing the effects of BASB034 polypeptideare most likely to be good antagonists. Molecules that bind well and, asthe case may be, increase the rate of product production from substrate,increase signal transduction, or increase chemical channel activity areagonists. Detection of the rate or level of, as the case may be,production of product from substrate, signal transduction, or chemicalchannel activity may be enhanced by using a reporter system. Reportersystems that may be useful in this regard include but are not limited tocolorimetric, labeled substrate converted into product, a reporter genethat is responsive to changes in BASB034 polynucleotide or polypeptideactivity, and binding assays known in the art.

[0115] Another example of an assay for BASB034 agonists is a competitiveassay that combines BASB034 and a potential agonist with BASB034-bindingmolecules, recombinant BASB034 binding molecules, natural substrates orligands, or substrate or ligand mimetics, under appropriate conditionsfor a competitive inhibition assay. BASB034 can be labeled, such as byradioactivity or a colorimetric compound, such that the number ofBASB034 molecules bound to a binding molecule or converted to productcan be determined accurately to assess the effectiveness of thepotential antagonist.

[0116] Potential antagonists include, among others, small organicmolecules, peptides, polypeptides and antibodies that bind to apolynucleotide and/or polypeptide of the invention and thereby inhibitor extinguish its activity or expression. Potential antagonists also maybe small organic molecules, a peptide, a polypeptide such as a closelyrelated protein or antibody that binds the same sites on a bindingmolecule, such as a binding molecule, without inducing BASB034-inducedactivities, thereby preventing the action or expression of BASB034polypeptides and/or polynucleotides by excluding BASB034 polypeptidesand/or polynucleotides from binding.

[0117] Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of BASB034.

[0118] In a further aspect, the present invention relates to geneticallyengineered soluble fusion proteins comprising a polypeptide of thepresent invention, or a fragment thereof, and various portions of theconstant regions of heavy or light chains of immunoglobulins of varioussubclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is theconstant part of the heavy chain of human IgG, particularly IgG1, wherefusion takes place at the hinge region. In a particular embodiment, theFc part can be removed simply by incorporation of a cleavage sequencewhich can be cleaved with blood clotting factor Xa. Furthermore, thisinvention relates to processes for the preparation of these fusionproteins by genetic engineering, and to the use thereof for drugscreening, diagnosis and therapy. A further aspect of the invention alsorelates to polynucleotides encoding such fusion proteins. Examples offusion protein technology can be found in International PatentApplication Nos. WO94/29458 and WO94/22914.

[0119] Each of the polynucleotide sequences provided herein may be usedin the discovery and development of antibacterial compounds. The encodedprotein, upon expression, can be used as a target for the screening ofantibacterial drugs. Additionally, the polynucleotide sequences encodingthe amino terminal regions of the encoded protein or Shine-Delgarno orother translation facilitating sequences of the respective mRNA can beused to construct antisense sequences to control the expression of thecoding sequence of interest.

[0120] The invention also provides the use of the polypeptide,polynucleotide, agonist or antagonist of the invention to interfere withthe initial physical interaction between a pathogen or pathogens and aeukaryotic, preferably mammalian, host responsible for sequelae ofinfection. In particular, the molecules of the invention may be used: inthe prevention of adhesion of bacteria, in particular gram positiveand/or gram negative bacteria, to eukaryotic, preferably mammalian,extracellular matrix proteins on in-dwelling devices or to extracellularmatrix proteins in wounds; to block bacterial adhesion betweeneukaryotic, preferably mammalian, extracellular matrix proteins andbacterial BASB034 proteins that mediate tissue damage and/or; to blockthe normal progression of pathogenesis in infections initiated otherthan by the implantation of in-dwelling devices or by other surgicaltechniques.

[0121] In accordance with yet another aspect of the invention, there areprovided BASB034 agonists and antagonists, preferably bacteristatic orbactericidal agonists and antagonists.

[0122] The antagonists and agonists of the invention may be employed,for instance, to prevent, inhibit and/or treat diseases.

[0123] In a further aspect, the present invention relates to mimotopesof the polypeptide of the invention. A mimotope is a peptide sequence,sufficiently similar to the native peptide (sequentially orstructurally), which is capable of being recognised by antibodies whichrecognise the native peptide; or is capable of raising antibodies whichrecognise the native peptide when coupled to a suitable carrier.

[0124] Peptide mimotopes may be designed for a particular purpose byaddition, deletion or substitution of elected amino acids. Thus, thepeptides may be modified for the purposes of ease of conjugation to aprotein carrier. For example, it may be desirable for some chemicalconjugation methods to include a terminal cysteine. In addition it maybe desirable for peptides conjugated to a protein carrier to include ahydrophobic terminus distal from the conjugated terminus of the peptide,such that the free unconjugated end of the peptide remains associatedwith the surface of the carrier protein. Thereby presenting the peptidein a conformation which most closely resembles that of the peptide asfound in the context of the whole native molecule. For example, thepeptides may be altered to have an N-terminal cysteine and a C-terminalhydrophobic amidated tail. Alternatively, the addition or substitutionof a D-stereoisomer form of one or more of the amino acids may beperformed to create a beneficial derivative, for example to enhancestability of the peptide.

[0125] Alternatively, peptide mimotopes may be identified usingantibodies which are capable themselves of binding to the polypeptidesof the present invention using techniques such as phage displaytechnology (EP 0 552 267 B1). This technique, generates a large numberof peptide sequences which mimic the structure of the native peptidesand are, therefore, capable of binding to anti-native peptideantibodies, but may not necessarily themselves share significantsequence homology to the native polypeptide.

[0126] Vaccines

[0127] Another aspect of the invention relates to a method for inducingan immunological response in an individual, particularly a mammal,preferably humans, which comprises inoculating the individual withBASB034 polynucleotide and/or polypeptide, or a fragment or variantthereof, adequate to produce antibody and/or T cell immune response toprotect said individual from infection, particularly bacterial infectionand most particularly Moraxella catarrhalis infection. Also provided aremethods whereby such immunological response slows bacterial replication.Yet another aspect of the invention relates to a method of inducingimmunological response in an individual which comprises delivering tosuch individual a nucleic acid vector, sequence or ribozyme to directexpression of BASB034 polynucleotide and/or polypeptide, or a fragmentor a variant thereof, for expressing BASB034 polynucleotide and/orpolypeptide, or a fragment or a variant thereof in vivo in order toinduce an immunological response, such as, to produce antibody and/or Tcell immune response, including, for example, cytokine-producing T cellsor cytotoxic T cells, to protect said individual, preferably a human,from disease, whether that disease is already established within theindividual or not. One example of administering the gene is byaccelerating it into the desired cells as a coating on particles orotherwise. Such nucleic acid vector may comprise DNA, RNA, a ribozyme, amodified nucleic acid, a DNA/RNA hybrid, a DNA-protein complex or anRNA-protein complex.

[0128] A further aspect of the invention relates to an immunologicalcomposition that when introduced into an individual, preferably a human,capable of having induced within it an immunological response, inducesan immunological response in such individual to a BASB034 polynucleotideand/or polypeptide encoded therefrom, wherein the composition comprisesa recombinant BASB034 polynucleotide and/or polypeptide encodedtherefrom and/or comprises DNA and/or RNA which encodes and expresses anantigen of said BASB034 polynucleotide, polypeptide encoded therefrom,or other polypeptide of the invention. The immunological response may beused therapeutically or prophylactically and may take the form ofantibody immunity and/or cellular immunity, such as cellular immunityarising from CTL or CD4+ T cells.

[0129] A BASB034 polypeptide or a fragment thereof may be fused withco-protein or chemical moiety which may or may not by itself produceantibodies, but which is capable of stabilizing the first protein andproducing a fused or modified protein which will have antigenic and/orimmunogenic properties, and preferably protective properties. Thus fusedrecombinant protein, preferably further comprises an antigenicco-protein, such as lipoprotein D from Haemophilus influenzae,Glutathione-S-transferase (GST) or beta-galactosidase, or any otherrelatively large co-protein which solubilizes the protein andfacilitates production and purification thereof. Moreover, theco-protein may act as an adjuvant in the sense of providing ageneralized stimulation of the immune system of the organism receivingthe protein. The co-protein may be attached to either the amino- orcarboxy-terminus of the first protein.

[0130] Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides and/orpolynucleotides of the invention and immunostimulatory DNA sequences,such as those described in Sato, Y. et al. Science 273: 352 (1996).

[0131] Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof, which have been shown toencode non-variable regions of bacterial cell surface proteins, inpolynucleotide constructs used in such genetic immunization experimentsin animal models of infection with Moraxella catarrhalis. Suchexperiments will be particularly useful for identifying protein epitopesable to provoke a prophylactic or therapeutic immune response. It isbelieved that this approach will allow for the subsequent preparation ofmonoclonal antibodies of particular value, derived from the requisiteorgan of the animal successfully resisting or clearing infection, forthe development of prophylactic agents or therapeutic treatments ofbacterial infection, particularly Moraxella catarrhalis infection, inmammals, particularly humans.

[0132] The invention also includes a vaccine formulation which comprisesan immunogenic recombinant polypeptide and/or polynucleotide of theinvention together with a suitable carrier, such as a pharmaceuticallyacceptable carrier. Since the polypeptides and polynucleotides may bebroken down in the stomach, each is preferably administeredparenterally, including, for example, administration that issubcutaneous, intramuscular, intravenous, or intradermal. Formulationssuitable for parenteral administration include aqueous and non-aqueoussterile injection solutions which may contain anti-oxidants, buffers,bacteriostatic compounds and solutes which render the formulationisotonic with the bodily fluid, preferably the blood, of the individual;and aqueous and non-aqueous sterile suspensions which may includesuspending agents or thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example, sealedampoules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use.

[0133] The vaccine formulation of the invention may also includeadjuvant systems for enhancing the immunogenicity of the formulation.Preferably the adjuvant system raises preferentially a TH1 type ofresponse.

[0134] An immune response may be broadly distinguished into two extremecatagories, being a humoral or cell mediated immune responses(traditionally characterised by antibody and cellular effectormechanisms of protection respectively). These categories of responsehave been termed TH1-type responses (cell-mediated response), andTH2-type immune responses (humoral response).

[0135] Extreme TH1-type immune responses may be characterised by thegeneration of antigen specific, haplotype restricted cytotoxic Tlymphocytes, and natural killer cell responses. In mice TH1-typeresponses are often characterised by the generation of antibodies of theIgG2a subtype, whilst in the human these correspond to IgG1 typeantibodies. TH2-type immune responses are characterised by thegeneration of a broad range of immunoglobulin isotypes including in miceIgG1, IgA, and IgM.

[0136] It can be considered that the driving force behind thedevelopment of these two types of immune responses are cytokines. Highlevels of TH1-type cytokines tend to favour the induction of cellmediated immune responses to the given antigen, whilst high levels ofTH2-type cytokines tend to favour the induction of humoral immuneresponses to the antigen.

[0137] The distinction of TH1 and TH2-type immune responses is notabsolute. In reality an individual will support an immune response whichis described as being predominantly TH1 or predominantly TH2. However,it is often convenient to consider the families of cytokines in terms ofthat described in murine CD4+ve T cell clones by Mosmann and Coffman(Mosmann, T. R. and Coffman, R. L. (1989) TH1 and TH2 cells: differentpatterns of lymphokine secretion lead to different functionalproperties. Annual Review of Immunology, 7, p145-173). Traditionally,TH1-type responses are associated with the production of the INF-γ andIL-2 cytokines by T-lymphocytes. Other cytokines often directlyassociated with the induction of TH1-type immune responses are notproduced by T-cells, such as IL-12. In contrast, TH2-type responses areassociated with the secretion of IL-4, IL-5, IL-6 and IL-13.

[0138] It is known that certain vaccine adjuvants are particularlysuited to the stimulation of either TH1 or TH2-type cytokine responses.Traditionally the best indicators of the TH1:TH2 balance of the immuneresponse after a vaccination or infection includes direct measurement ofthe production of TH1 or TH2 cytokines by T lymphocytes in vitro afterrestimulation with antigen, and/or the measurement of the IgG1:IgG2aratio of antigen specific antibody responses.

[0139] Thus, a TH1-type adjuvant is one which preferentially stimulatesisolated T-cell populations to produce high levels of TH1-type cytokineswhen re-stimulated with antigen in vitro, and promotes development ofboth CD8+cytotoxic T lymphocytes and antigen specific immunoglobulinresponses associated with TH1-type isotype.

[0140] Adjuvants which are capable of preferential stimulation of theTH1 cell response are described in International Patent Application No.WO 94/00153 and WO 95/17209.

[0141] 3 De-O-acylated monophosphoryl lipid A (3D-MPL) is one suchadjuvant. This is known from GB 2220211 (Ribi). Chemically it is amixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6acylated chains and is manufactured by Ribi Immunochem, Montana. Apreferred form of 3 De-O-acylated monophosphoryl lipid A is disclosed inEuropean Patent 0 689 454 B1 (SmithKline Beecham Biologicals SA).

[0142] Preferably, the particles of 3D-MPL are small enough to besterile filtered through a 0.22 micron membrane (European Patent number0 689 454). 3D-MPL will be present in the range of 10 μg-100 μgpreferably 25-50 μg per dose wherein the antigen will typically bepresent in a range 2-50 μg per dose.

[0143] Another preferred adjuvant comprises QS21, an Hplc purifiednon-toxic fraction derived from the bark of Quillaja Saponaria Molina.Optionally this may be admixed with 3 De-O-acylated monophosphoryl lipidA (3D-MPL), optionally together with an carrier.

[0144] The method of production of QS21 is disclosed in U.S. Pat. No.5,057,540.

[0145] Non-reactogenic adjuvant formulations containing QS21 have beendescribed previously (WO 96/33739). Such formulations comprising QS21and cholesterol have been shown to be successful TH1 stimulatingadjuvants when formulated together with an antigen.

[0146] Further adjuvants which are preferential stimulators of TH1 cellresponse include immunomodulatory oligonucleotides, for exampleunmethylated CpG sequences as disclosed in WO 96/02555.

[0147] Combinations of different TH1 stimulating adjuvants, such asthose mentioned hereinabove, are also contemplated as providing anadjuvant which is a preferential stimulator of TH1 cell response. Forexample, QS21 can be formulated together with 3D-MPL. The ratio ofQS21:3D-MPL will typically be in the order of 1:10 to 10:1; preferably1:5 to 5:1 and often substantially 1:1. The preferred range for optimalsynergy is 2.5:1 to 1:1 3D-MPL: QS21.

[0148] Preferably a carrier is also present in the vaccine compositionaccording to the invention. The carrier may be an oil in water emulsion,or an aluminium salt, such as aluminium phosphate or aluminiumhydroxide.

[0149] A preferred oil-in-water emulsion comprises a metabolisible oil,such as squalene, alpha tocopherol and Tween 80. In a particularlypreferred aspect the antigens in the vaccine composition according tothe invention are combined with QS21 and 3D-MPL in such an emulsion.Additionally the oil in water emulsion may contain span 85 and/orlecithin and/or tricaprylin.

[0150] Typically for human administration QS21 and 3D-MPL will bepresent in a vaccine in the range of 1 μg-200 μg, such as 10-100 g,preferably 10 μg-50 μg per dose. Typically the oil in water willcomprise from 2 to 10% squalene, from 2 to 10% alpha tocopherol and from0.3 to 3% tween 80. Preferably the ratio of squalene: alpha tocopherolis equal to or less than 1 as this provides a more stable emulsion. Span85 may also be present at a level of 1%. In some cases it may beadvantageous that the vaccines of the present invention will furthercontain a stabiliser.

[0151] Non-toxic oil in water emulsions preferably contain a non-toxicoil, e.g. squalane or squalene, an emulsifier, e.g. Tween 80, in anaqueous carrier. The aqueous carrier may be, for example, phosphatebuffered saline.

[0152] A particularly potent adjuvant formulation involving QS21, 3D-MPLand tocopherol in an oil in water emulsion is described in WO 95/17210.

[0153] The present invention also provides a polyvalent vaccinecomposition comprising a vaccine formulation of the invention incombination with other antigens, in particular antigens useful fortreating cancers, autoimmune diseases and related conditions. Such apolyvalent vaccine composition may include a TH-1 inducing adjuvant ashereinbefore described.

[0154] While the invention has been described with reference to certainBASB034 polypeptides and polynucleotides, it is to be understood thatthis covers fragments of the naturally occurring polypeptides andpolynucleotides, and similar polypeptides and polynucleotides withadditions, deletions or substitutions which do not substantially affectthe immunogenic properties of the recombinant polypeptides orpolynucleotides.

[0155] Compositions, Kits and Administration

[0156] In a further aspect of the invention there are providedcompositions comprising a BASB034 polynucleotide and/or a BASB034polypeptide for administration to a cell or to a multicellular organism.

[0157] The invention also relates to compositions comprising apolynucleotide and/or a polypeptides discussed herein or their agonistsor antagonists. The polypeptides and polynucleotides of the inventionmay be employed in combination with a non-sterile or sterile carrier orcarriers for use with cells, tissues or organisms, such as apharmaceutical carrier suitable for administration to an individual.Such compositions comprise, for instance, a media additive or atherapeutically effective amount of a polypeptide and/or polynucleotideof the invention and a pharmaceutically acceptable carrier or excipient.Such carriers may include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol and combinations thereof. Theformulation should suit the mode of administration. The inventionfurther relates to diagnostic and pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

[0158] Polypeptides, polynucleotides and other compounds of theinvention may be employed alone or in conjunction with other compounds,such as therapeutic compounds.

[0159] The pharmaceutical compositions may be administered in anyeffective, convenient manner including, for instance, administration bytopical, oral, anal, vaginal, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes amongothers.

[0160] In therapy or as a prophylactic, the active agent may beadministered to an individual as an injectable composition, for exampleas a sterile aqueous dispersion, preferably isotonic.

[0161] In a further aspect, the present invention provides forpharmaceutical compositions comprising a therapeutically effectiveamount of a polypeptide and/or polynucleotide, such as the soluble formof a polypeptide and/or polynucleotide of the present invention, agonistor antagonist peptide or small molecule compound, in combination with apharmaceutically acceptable carrier or excipient. Such carriers include,but are not limited to, saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The invention furtherrelates to pharmaceutical packs and kits comprising one or morecontainers filled with one or more of the ingredients of theaforementioned compositions of the invention. Polypeptides,polynucleotides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

[0162] The composition will be adapted to the route of administration,for instance by a systemic or an oral route. Preferred forms of systemicadministration include injection, typically by intravenous injection.Other injection routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if a polypeptide or other compounds of the present inventioncan be formulated in an enteric or an encapsulated formulation, oraladministration may also be possible. Administration of these compoundsmay also be topical and/or localized, in the form of salves, pastes,gels, solutions, powders and the like.

[0163] For administration to mammals, and particularly humans, it isexpected that the daily dosage level of the active agent will be from0.01 mg/kg to 10 mg/kg, typically around 1 mg/kg. The physician in anyevent will determine the actual dosage which will be most suitable foran individual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

[0164] The dosage range required depends on the choice of peptide, theroute of administration, the nature of the formulation, the nature ofthe subject's condition, and the judgment of the attending practitioner.Suitable dosages, however, are in the range of 0.1-100 μg/kg of subject.

[0165] A vaccine composition is conveniently in injectable form.Conventional adjuvants may be employed to enhance the immune response. Asuitable unit dose for vaccination is 0.5-5 microgram/kg of antigen, andsuch dose is preferably administered 1-3 times and with an interval of1-3 weeks. With the indicated dose range, no adverse toxicologicaleffects will be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

[0166] Wide variations in the needed dosage, however, are to be expectedin view of the variety of compounds available and the differingefficiencies of various routes of administration. For example, oraladministration would be expected to require higher dosages thanadministration by intravenous injection. Variations in these dosagelevels can be adjusted using standard empirical routines foroptimization, as is well understood in the art.

[0167] Sequence Databases, Sequences in a Tangible Medium, andAlgorithms

[0168] Polynucleotide and polypeptide sequences form a valuableinformation resource with which to determine their 2- and 3-dimensionalstructures as well as to identify further sequences of similar homology.These approaches are most easily facilitated by storing the sequence ina computer readable medium and then using the stored data in a knownmacromolecular structure program or to search a sequence database usingwell known searching tools, such as the GCG program package.

[0169] Also provided by the invention are methods for the analysis ofcharacter sequences or strings, particularly genetic sequences orencoded protein sequences. Preferred methods of sequence analysisinclude, for example, methods of sequence homology analysis, such asidentity and similarity analysis, DNA, RNA and protein structureanalysis, sequence assembly, cladistic analysis, sequence motifanalysis, open reading frame determination, nucleic acid base calling,codon usage analysis, nucleic acid base trimming, and sequencingchromatogram peak analysis.

[0170] A computer based method is provided for performing homologyidentification. This method comprises the steps of: providing a firstpolynucleotide sequence comprising the sequence of a polynucleotide ofthe invention in a computer readable medium; and comparing said firstpolynucleotide sequence to at least one second polynucleotide orpolypeptide sequence to identify homology.

[0171] A computer based method is also provided for performing homologyidentification, said method comprising the steps of: providing a firstpolypeptide sequence comprising the sequence of a polypeptide of theinvention in a computer readable medium; and comparing said firstpolypeptide sequence to at least one second polynucleotide orpolypeptide sequence to identify homology.

[0172] All publications and references, including but not limited topatents and patent applications, cited in this specification are hereinincorporated by reference in their entirety as if each individualpublication or reference were specifically and individually indicated tobe incorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

[0173] Definitions

[0174] “Identity,” as known in the art, is a relationship between two ormore polypeptide sequences or two or more polynucleotide sequences, asthe case may be, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”can be readily calculated by known methods, including but not limited tothose described in (Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heine, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math.,48: 1073 (1988). Methods to determine identity are designed to give thelargest match between the sequences tested. Moreover, methods todetermine identity are codified in publicly available computer programs.Computer program methods to determine identity between two sequencesinclude, but are not limited to, the GAP program in the GCG programpackage (Devereux, J., et al., Nucleic Acids Research 12(1): 387(1984)), BLASTP, BLASTN (Altschul, S. F. et al., J. Molec. Biol. 215:403-410 (1990), and FASTA (Pearson and Lipman Proc. Natl. Acad. Sci. USA85; 2444-2448 (1988). The BLAST family of programs is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). The well known Smith Waterman algorithm may also be usedto determine identity.

[0175] Parameters for polypeptide sequence comparison include thefollowing:

[0176] Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

[0177] Comparison matrix: BLOSSUM62 from Henikoff and Henikoff,

[0178] Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992)

[0179] Gap Penalty: 8

[0180] Gap Length Penalty: 2

[0181] A program useful with these parameters is publicly available asthe “gap” program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

[0182] Parameters for polynucleotide comparison include the following:

[0183] Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

[0184] Comparison matrix: matches=+10, mismatch=0

[0185] Gap Penalty: 50

[0186] Gap Length Penalty: 3

[0187] Available as: The “gap” program from Genetics Computer Group,Madison Wis. These are the default parameters for nucleic acidcomparisons.

[0188] A preferred meaning for “identity” for polynucleotides andpolypeptides, as the case may be, are provided in (1) and (2) below.

[0189] (1) Polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide sequence having at least a50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the referencesequence of SEQ ID NO:1, wherein said polynucleotide sequence may beidentical to the reference sequence of SEQ ID NO:1 or may include up toa certain integer number of nucleotide alterations as compared to thereference sequence, wherein said alterations are selected from the groupconsisting of at least one nucleotide deletion, substitution, includingtransition and transversion, or insertion, and wherein said alterationsmay occur at the 5′ or 3′ terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among the nucleotides in the reference sequence orin one or more contiguous groups within the reference sequence, andwherein said number of nucleotide alterations is determined bymultiplying the total number of nucleotides in SEQ ID NO:1 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of nucleotides in SEQ IDNO:1, or:

n _(n) ≦x _(n)−(x _(n) ·y),

[0190] wherein n_(n) is the number of nucleotide alterations, x_(n) isthe total number of nucleotides in SEQ ID NO:1, y is 0.50 for 50%, 0.60for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95for 95%, 0.97 for 97% or 1.00 for 100%, and · is the symbol for themultiplication operator, and wherein any non-integer product of x_(n)and y is rounded down to the nearest integer prior to subtracting itfrom x_(n). Alterations of a polynucleotide sequence encoding thepolypeptide of SEQ ID NO:2 may create nonsense, missense or frameshiftmutations in this coding sequence and thereby alter the polypeptideencoded by the polynucleotide following such alterations.

[0191] By way of example, a polynucleotide sequence of the presentinvention may be identical to the reference sequence of SEQ ID NO:1,that is it may be 100% identical, or it may include up to a certaininteger number of nucleic acid alterations as compared to the referencesequence such that the percent identity is less than 100% identity. Suchalterations are selected from the group consisting of at least onenucleic acid deletion, substitution, including transition andtransversion, or insertion, and wherein said alterations may occur atthe 5′ or 3′ terminal positions of the reference polynucleotide sequenceor anywhere between those terminal positions, interspersed eitherindividually among the nucleic acids in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofnucleic acid alterations for a given percent identity is determined bymultiplying the total number of nucleic acids in SEQ ID NO:1 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of nucleic acids in SEQID NO:1, or:

n _(n) ≦x _(n)−(x _(n) ·y),

[0192] wherein n_(n) is the number of nucleic acid alterations, x_(n) isthe total number of nucleic acids in SEQ ID NO:1, y is, for instance0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., · is the symbol for themultiplication operator, and wherein any non-integer product of x_(n)and y is rounded down to the nearest integer prior to subtracting itfrom x_(n).

[0193] (2) Polypeptide embodiments further include an isolatedpolypeptide comprising a polypeptide having at least a 50, 60, 70, 80,85, 90, 95, 97 or 100% identity to a polypeptide reference sequence ofSEQ ID NO:2, wherein said polypeptide sequence may be identical to thereference sequence of SEQ ID NO:2 or may include up to a certain integernumber of amino acid alterations as compared to the reference sequence,wherein said alterations are selected from the group consisting of atleast one amino acid deletion, substitution, including conservative andnon-conservative substitution, or insertion, and wherein saidalterations may occur at the amino- or carboxy-terminal positions of thereference polypeptide sequence or anywhere between those terminalpositions, interspersed either individually among the amino acids in thereference sequence or in one or more contiguous groups within thereference sequence, and wherein said number of amino acid alterations isdetermined by multiplying the total number of amino acids in SEQ ID NO:2by the integer defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO:2, or:

n _(a) ≦x _(a)−(x _(a) ·y),

[0194] wherein n_(a) is the number of amino acid alterations, x_(a) isthe total number of amino acids in SEQ ID NO:2, y is 0.50 for 50%, 0.60for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95for 95%, 0.97 for 97% or 1.00 for 100%, and · is the symbol for themultiplication operator, and wherein any non-integer product of x_(a)and y is rounded down to the nearest integer prior to subtracting itfrom x_(a).

[0195] By way of example, a polypeptide sequence of the presentinvention may be identical to the reference sequence of SEQ ID NO:2,that is it may be 100% identical, or it may include up to a certaininteger number of amino acid alterations as compared to the referencesequence such that the percent identity is less than 100% identity. Suchalterations are selected from the group consisting of at least one aminoacid deletion, substitution, including conservative and non-conservativesubstitution, or insertion, and wherein said alterations may occur atthe amino- or carboxy-terminal positions of the reference polypeptidesequence or anywhere between those terminal positions, interspersedeither individually among the amino acids in the reference sequence orin one or more contiguous groups within the reference sequence. Thenumber of amino acid alterations for a given % identity is determined bymultiplying the total number of amino acids in SEQ ID NO:2 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO:2, or:

n _(a) ≦x _(a)−(x _(a) ·y),

[0196] wherein n_(a) is the number of amino acid alterations, x_(a) isthe total number of amino acids in SEQ ID NO:2, y is, for instance 0.70for 70%, 0.80 for 80%, 0.85 for 85% etc., and · is the symbol for themultiplication operator, and wherein any non-integer product of x_(a)and y is rounded down to the nearest integer prior to subtracting itfrom x_(a).

[0197] “Individual(s),” when used herein with reference to an organism,means a multicellular eukaryote, including, but not limited to ametazoan, a mammal, an ovid, a bovid, a simian, a primate, and a human.

[0198] “Isolated” means altered “by the hand of man” from its naturalstate, i.e., if it occurs in nature, it has been changed or removed fromits original environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein. Moreover, a polynucleotide or polypeptide that is introducedinto an organism by transformation, genetic manipulation or by any otherrecombinant method is “isolated” even if it is still present in saidorganism, which organism may be living or non-living.

[0199] “Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA including single and double-stranded regions.

[0200] “Variant” refers to a polynucleotide or polypeptide that differsfrom a reference polynucleotide or polypeptide, but retains essentialproperties. A typical variant of a polynucleotide differs in nucleotidesequence from another, reference polynucleotide. Changes in thenucleotide sequence of the variant may or may not alter the amino acidsequence of a polypeptide encoded by the reference polynucleotide.Nucleotide changes may result in amino acid substitutions, additions,deletions, fusions and truncations in the polypeptide encoded by thereference sequence, as discussed below. A typical variant of apolypeptide differs in amino acid sequence from another, referencepolypeptide. Generally, differences are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overalland, in many regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more substitutions, additions,deletions in any combination. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code. A variant ofa polynucleotide or polypeptide may be a naturally occurring such as anallelic variant, or it may be a variant that is not known to occurnaturally. Non-naturally occurring variants of polynucleotides andpolypeptides may be made by mutagenesis techniques or by directsynthesis.

[0201] “Disease(s)” means any disease caused by or related to infectionby a bacteria, including, for example, otitis media in infants andchildren, pneumonia in elderlies, sinusitis, nosocomial infections andinvasive diseases, chronic otitis media with hearing loss, fluidaccumulation in the middle ear, auditive nerve damage, delayed speechlearning, infection of the upper respiratory tract and inflammation ofthe middle ear.

EXAMPLES

[0202] The examples below are carried out using standard techniques,which are well known and routine to those of skill in the art, exceptwhere otherwise described in detail. The examples are illustrative, butdo not limit the invention.

Example 1

[0203] Discovery and Confirmatory DNA Sequencing of the BASB034 Genefrom Moraxella catarrhalis Strain ATCC 43617.

[0204] The BASB034 gene was first discovered in the Incyte PathoSeq database containing unfinished genomic DNA sequences of the Moraxellacatarrhalis strain ATCC 43617 (also referred to as strain MC2931). Thetranslation of the BASB034 polynucleotide sequence showed significantsimilarity (42% identity in a 214 amino acids overlap) to a Klebsiellapneumoniae outer membrane phospholipase A protein.

[0205] The sequence of the BASB034 gene was further confirmedexperimentally. For this purpose, genomic DNA was extracted from 10¹⁰cells of the M. catarrhalis cells (strain ATCC 43617) using the QIAGENgenomic DNA extraction kit (Qiagen Gmbh), and 1 μg of this material wassubmitted to Polymerase Chain Reaction DNA amplification using primersE481124 (5′-GAT TTA AGA GTA TGT TAT GAT G-3′) [SEQ ID NO:9] and E481125(5′-GTA TGG GTT GAT CAA ATA CAG-3′) [SEQ ID NO:10]. This PCR product waspurified on a Biorobot 9600 (Qiagen Gmbh) apparatus and subjected to DNAsequencing using the Big Dye Cycle Sequencing kit (Perkin-Elmer) and anABI 377/PRISM DNA sequencer. DNA sequencing was performed on bothstrands with a redundancy of 2 and the full-length sequence wasassembled using the Sequencher™ software (Applied Biosytems). Theresulting DNA sequence turned out to be 100% identical to SEQ ID NO:1.

Example 2

[0206] Variability Analysis of the BASB034 Gene Among Several Moraxellacatarrhalis Strains.

[0207] 2A: Restriction Fragment Length Analysis (RFLP).

[0208] Genomic DNA was extracted from 16 M. catarrhalis strains(presented in Table 1) as described below. M. catarrhalis was streakedfor single colonies on BHI agar plates and grown overnight at 37° C.Three or four single colonies were picked and used to inoculate a ˜1.5ml BHI (Brain-heart infusion) broth seed culture which was grownovernight in a shaking incubator, ˜300 rpm, at 37° C. A 500 mlerlenmeyer flask containing ˜150 ml of BHI broth was inoculated with theseed culture and grown for ˜12-16 hours at 37° C. in a shakingincubator, ˜175 rpm, to generate cell mass for DNA isolation. Cells werecollected by centrifugation in a Sorvall GSA rotor at ˜2000×g for 15minutes at room temperature. The supernatant was removed and the cellpellet suspended in ˜5.0 ml of sterile water. An equal volume of lysisbuffer (200 mM NaCl, 20 mM EDTA, 40 mM Tris-Hcl, pH 8.0, 0.5% (w/v) SDS,0.5% (v/v) 2-mercaptoethanol, and 250 μg/ml of proteinase K) was addedand the cells suspended by gentle agitation and trituration. The cellsuspension was then incubated ˜12 hours at 50° C. to lyse the bacteriaand liberate chromosomal DNA. Proteinaceous material was precipitated bythe addition of 5.0 ml of saturated NaCl (˜6.0 M, in sterile water) andcentrifugation at ˜5,500×g in a Sorvall SS34 rotor at room temperature.Chromosomal DNA was precipitated from the cleared supernatant by theaddition of two volumes of 100% ethanol. Aggregated DNA was collectedand washed using gentle agitation in a small volume of a 70% ethanolsolution. Purified chromosomal DNA was suspended in sterile water andallowed to dissolve/disburse overnight at 4° C. by gentle rocking. Theconcentration of dissolved DNA was determined spectrophotometrically at260 nm using an extinction coefficient of 1.0 O.D. unit ˜50 μg/ml.

[0209] This material was next submitted to PCR amplification using theMC-Pla-BamF (5′-AAG GGC CCA ATT ACG CAG AGG GGA TCC CAA GCT GTA CCA AATCCT GTG GCA TTT GTT G-3′) [SEQ ID NO:11] and MC-Pla-SaLRC (5′-AAG GGCCCA ATT ACG CAG AGG GTC GAC TTA TTA TAG ACC CAT CCA GTC GTT AAG CATAAG-3′) [SEQ ID NO:12] oligonucleotides. The corresponding BASB034 geneamplicons were then subjected independently to hydrolysis usingrestriction enzymes (HphI, AluI, Rsa I, EcoRV, Sau3AI) and restrictionproducts were separated by agarose or polyacrylamide gel electrophoresisusing standard molecular biology procedures as described in “MolecularCloning, a Laboratory Manual, Second Edition, Eds: Sambrook, Fritsch &Maniatis, Cold Spring Harbor press 1989”. The photographs of theresulting electrophoresis gels are displayed in FIG. 1. For each strain,RFLP patterns corresponding to the 6 restriction enzymes were scored andcombined. Groups of strains sharing identical combination of RFLPpatterns were then defined. Using this methodology, the strains testedin this study fell into 3 genomic groups (Group 1: Mc2931, Mc 2904, Mc2905, Mc2907, Mc2909, Mc2910, Mc2911, Mc2912, Mc2926, Mc2931, Mc2956,Mc2969, Mc2975; Group 2: Mc2906, Mc2913; Group 3: Mc2908. (Mc2960 failedto be amplified and consequently was not classified). These data supportthat the Moraxella catarrhalis population used in this study displayslimited nucleotide sequence diversity for the BASB034 gene.

[0210] 2B: DNA Sequencing in Other Strains.

[0211] Using the experimental procedure described in Example 1, thesequence of the BASB034 gene was also determined for three additionalMoraxella catarrhalis strains. The nucleotide sequences of the BASB034gene of the strains Mc2908, Mc2913 and Mc2969, representative of thethree genomic groups identified previously, are shown in SEQ ID NO:3, 5and 7, respectively. These nucleotide sequences were translated intoamino acid sequences, which are shown in SEQ ID NO:4, 6 and 8,respectively. Using the MegAlign program from the DNASTAR Lasergenepackage, a multiple alignment of the nucleotide sequences of SEQ IDNO:1, 3, 5 and 7 was performed, and is displayed in FIG. 2. A pairwisecomparison of identities is summarized in Table 2, showing that the fourBASB034 nucleotide gene sequences are all similar at an identity levelgreater than 98%. Using the same program, a multiple alignment of theprotein sequences of SEQ ID NO:2, 4, 6 and 8 was performed, and isdisplayed in FIG. 3. A pairwise comparison of identities is summarizedin Table 3, showing that the four BASB034 protein sequences are allsimilar at an identity level greater than 98%. Taken together, thesedata indicate very strong sequence conservation of the BASB034 geneamong Moraxella catarrhalis strains. TABLE 1 Features of the Moraxellacatarrhalis strains used in this study Strain Isolated in: from: Mc2904USA Tympanocentesis Mc2905 USA Tympanocentesis Mc2906 USATympanocentesis Mc2907 USA Tympanocentesis Mc2908 USA Acute otitisTympanocentesis Mc2909 USA Tympanocentesis Mc2910 USA TympanocentesisMc2911 USA Acute otitis Tympanocentesis Mc2912 USA Acute otitisTympanocentesis Mc2913 USA Acute otitis Tympanocentesis Mc2926 USATympanocentesis Mc2931 USA Transtracheal aspirate /ATCC 43617 Mc2956Finland Middle ear fluid Mc2960 Finland Middle ear fluid Mc2969 NorwayNasopharynx (Pharyngitis-Rhinitis) Mc2975 Norway Nasopharynx (Rhinitis)

[0212] TABLE 2 Pairwise identities of the BASB034 polynucleotidesequences (in %) SeqID No:3 SeqID No:5 SeqID No:7 SeqID No:1 98.7 99.299.7 SeqID No:3 99.3 98.7 SeqID No:5 99.1

[0213] TABLE 3 Pairwise identities of the BASB034 polypeptide sequences(in %) SeqID No:4 SeqID No:6 SeqID No:8 SeqID No:2 98.6 99.3 99.5 SeqIDNo:4 98.9 99.1 SeqID No:6 99.3

Example 3 Construction of Plasmid to Express Recombinant BASB034

[0214] A: Cloning of BASB034.

[0215] The BamHI and SalI restriction sites engineered into the forward([SEQ ID NO:11]) and reverse ([SEQ ID NO:12]) amplification primers,respectively, permitted directional cloning of a BASB034 PCR productinto the commercially available E. coli expression plasmid pQE30(QiaGen, ampicillin resistant) such that a mature BASB034 protein couldbe expressed as a fusion protein containing a (His)6 affinitychromatography tag at the N-terminus. The BASB034 PCR product waspurified from the amplification reaction using silica gel-based spincolumns (QiaGen) according to the manufacturers instructions. To producethe required BamHI and SalI termini necessary for cloning, purified PCRproduct was sequentially digested to completion with BamHI and SalIrestriction enzymes as recommended by the manufacturer (LifeTechnologies). Following the first restriction digestion, the PCRproduct was purified via spin column as above to remove salts and elutedin sterile water prior to the second enzyme digestion. The digested DNAfragment was again purified using silica gel-based spin columns prior toligation with the pQE30 plasmid.

[0216] B: Production of Expression Vector.

[0217] To prepare the expression plasmid pQE30 for ligation, it wassimilarly digested to completion with both BamHI and SalI and thentreated with calf intestinal phosphatase (CIP, ˜0.02 units/pmole of 5′end, Life Technologies) as directed by the manufacturer to prevent selfligation. An approximately 5-fold molar excess of the digested fragmentto the prepared vector was used to program the ligation reaction. Astandard ˜20 μl ligation reaction (˜16° C., ˜16 hours), using methodswell known in the art, was performed using T4 DNA ligase (˜2.0units/reaction, Life Technologies). An aliquot of the ligation (˜5 μl)was used to transform electro-competent M15 (pREP4) cells according tomethods well known in the art. Following a ˜2-3 hour outgrowth period at37° C. in ˜1.0 ml of LB broth, transformed cells were plated on LB agarplates containing kanamycin (50 μg/ml) and ampicillin (100 μg/ml). Bothantibiotics were included in the selection media to ensure that alltransformed cells carried both the pREP4 plasmid (KnR), which carriesthe lacIq gene necessary for the repression of expression forIPTG-inducible expression of proteins on pQE30, and the pQE30-BASB034plasmid (ApR). Plates were incubated overnight at 37° C. for ˜16 hours.Individual KnR/ApR colonies were picked with sterile toothpicks and usedto “patch” inoculate fresh LB KnR/ApR plates as well as a ˜1.0 ml LBKnR/ApR broth culture. Both the patch plates and the broth culture wereincubated overnight at 37° C. in either a standard incubator (plates) ora shaking water bath.

[0218] A whole cell-based PCR analysis was employed to verify thattransformants contained the BASB034 DNA insert. Here, the ˜1.0 mlovernight LB Kn/Ap broth culture was transferred to a 1.5 mlpolypropylene tube and the cells collected by centrifugation in aBeckman microcentrifuge (˜3 min., room temperature, ˜12,000×g). The cellpellet was suspended in ˜200 μl of sterile water and a ˜10 μl aliquotused to program a ˜50 μl final volume PCR reaction containing bothBASB034 forward and reverse amplification primers. Final concentrationsof the PCR reaction components were essentially the same as thosespecified in example 2 except ˜5.0 units of Taq polymerase was used. Theinitial 95° C. denaturation step was increased to 3 minutes to ensurethermal disruption of the bacterial cells and liberation of plasmid DNA.An ABI Model 9700 thermal cycler and a 32 cycle, three-step thermalamplification profile, i.e. 95° C., 45 sec; 55-58° C., 45 sec, 72° C., 1min., were used to amplify the BASB034 PCR fragment from the lysedtransformant samples. Following thermal amplification, a ˜20 μl aliquotof the reaction was analyzed by agarose gel electrophoresis (0.8%agarose in a Tris-acetate-EDTA (TAE) buffer). DNA fragments werevisualized by UV illumination after gel electrophoresis and ethidiumbromide staining. A DNA molecular size standard (1 Kb ladder, LifeTechnologies) was electrophoresed in parallel with the test samples andwas used to estimate the size of the PCR products. Transformants thatproduced the expected PCR product were identified as strains containinga BASB034 expression construct. Expression plasmid containing strainswere then analyzed for the inducible expression of recombinant BASB034.

[0219] C: Expression Analysis of PCR-Positive Transformants.

[0220] For each PCR-positive transformant identified above, ˜5.0 ml ofLB broth containing kanamycin (50 μg/ml) and ampicillin (100 μg/ml) wasinoculated with cells from the patch plate and grown overnight at 37° C.with shaking (˜250 rpm). An aliquot of the overnight seed culture (˜1.0ml) was inoculated into a 125 ml erlenmeyer flask containing ˜25 ml ofLB Kn/Ap broth and grown at 37° C. with shaking (˜250 rpm) until theculture turbidity reached O.D.600 of ˜0.5, i.e. mid-log phase (usuallyabout 1.5-2.0 hours). At this time approximately half of the culture(˜12.5 ml) was transferred to a second 125 ml flask and expression ofrecombinant BASB034 protein induced by the addition of IPTG (1.0 M stockprepared in sterile water, Sigma) to a final concentration of 1.0 mM.Incubation of both the IPTG-induced and non-induced cultures continuedfor an additional 4 hours at 37° C. with shaking. Samples (˜1.0 ml) ofboth induced and non-induced cultures were removed after the inductionperiod and the cells collected by centrifugation in a microcentrifuge atroom temperature for ˜3 minutes. Individual cell pellets were suspendedin ˜50 μl of sterile water, then mixed with an equal volume of 2×Laemelli SDS-PAGE sample buffer containing 2-mercaptoethanol, and placedin boiling water bath for ˜3 min to denature protein. Equal volumes (˜15μl) of both the crude IPTG-induced and the non-induced cell lysates wereloaded onto duplicate 12% Tris/glycine polyacrylamide gel (1 mm thickMini-gels, Novex). The induced and non-induced lysate samples wereelectrophoresed together with prestained molecular weight markers(SeeBlue, Novex) under conventional conditions using a standardSDS/Tris/glycine running buffer (BioRad). Following electrophoresis, onegel was stained with commassie brilliant blue R250 (BioRad) and thendestained to visualize novel BASB034 IPTG-inducible protein(s). Thesecond gel was electroblotted onto a PVDF membrane (0.45 micron poresize, Novex) for 2 hrs at 4° C. using a BioRad Mini-Protean II blottingapparatus and Towbin's methanol (20%) transfer buffer. Blocking of themembrane and antibody incubations were performed according to methodswell known in the art. A monoclonal anti-RGS (His)3 antibody, followedby a second rabbit anti-mouse antibody conjugated to HRP (QiaGen), wasused to confirm the expression and identity of the BASB034 recombinantprotein. Visualization of the anti-His antibody reactive pattern wasachieved using either an ABT insoluble substrate or using Hyperfilm withthe Amersham ECL chemiluminescence system.

[0221] D: Sequence Confirmation.

[0222] To further verify that the IPTG-inducible recombinant BASB034protein being expressed is in the correct open reading frame and not aspurious molecule arising from a cloning artifact (i.e. a frame-shift),the DNA sequence of the cloned insert was determined. The DNA sequencefor the M. catarrhalis BASB034 gene was obtained from one strand usingconventional asymmetric PCR cycle sequencing methodologies (ABI PrismDye-Terminator Cycle Sequencing, Perkin-Elmer). Sequencing reactionswere programmed with undigested expression plasmid DNA (˜0.5 μg/rxn) asa template and appropriate pQE30 vector-specific and ORF-specificsequencing primers (˜3.5 pmol/rxn). In addition to the template andsequencing primer, each sequencing reaction (˜20 μl) contained the fourdifferent dNTPs (i.e. A, G, C, and T) and the four corresponding ddNTPs(i.e. ddA, ddG, ddC, and ddT) terminator nucleotides; with eachterminator being conjugated to one of the four fluorescent dyes, Joe,Tam, Rox, or Fam. Single strand sequencing elongation products wereterminated at random positions along the template by the incorporationof the dye-labelled ddNTP terminators. Fluorescent dye-labelledtermination products were purified using microcentrifuge size-exclusionchromatography columns (Princeton Genetics), dried under vacuum,suspended in a Template Resuspension Buffer (Perkin-Elmer) for capillaryelectrophoresis or deionized formamide for PAGE, denatured at 95° C. for˜5 min, and analyzed by high resolution capillary electrophoresis (ABI310 Automated DNA Sequenator, Perkin-Elmer) or high resolution PAGE (ABI377 Automated DNA Sequenator) as recommended by the manufacturer. DNAsequence data produced from individual reactions were collected and therelative fluorescent peak intensities analyzed automatically on aPowerMAC computer using ABI Sequence Analysis Software (Perkin-Elmer).Individually autoanalyzed DNA sequences were edited manually foraccuracy before being merged into a consensus single strand sequence“string” using AutoAssembler software (Perkin-Elmer). Sequencingdetermined that the expression plasmid contained the correct sequence inthe correct open reading frame.

Example 4 Production of Recombinant BASB034

[0223] Bacterial Strain

[0224] A recombinant expression strain of E. coli M15 (pREP4) containinga plasmid (pQE30) encoding BASB034 from M. catarrhalis. was used toproduce cell mass for purification of recombinant protein. Theexpression strain was cultivated on LB agar plates containing 50 μg/mlkanamycin (“Kn”) and 100 μg/ml ampicillin (“Ap”) to ensure both thepREP4 lacIq control plasmid and the pQE30-BASB034 expression constructwere both maintained. For cryopreservation at −80° C., the strain waspropagated in LB broth containing the same concentration of antibioticsthen mixed with an equal volume of LB broth containing 30% (w/v)glycerol.

[0225] Media

[0226] The fermentation medium used for the production of recombinantprotein consisted of 2×YT broth (Difco) containing 50 μg/ml Kn and 100μg/ml Ap. Antifoam was added to medium for the fermentor at 0.25 ml/L(Antifoam 204, Sigma). To induce expression of the BASB034 recombinantprotein, IPTG (Isopropyl β-D-Thiogalactopyranoside) was added to thefermentor (1 mM, final).

[0227] Fermentation

[0228] A 500-ml erlenmeyer seed flask, containing 50 ml working volume,was inoculated with 0.3 ml of rapidly thawed frozen culture, or severalcolonies from a selective agar plate culture, and incubated forapproximately 12 hours at 37 ±1° C. on a shaking platform at 150 rpm(Innova 2100, New Brunswick Scientific). This seed culture was then usedto inoculate a 5-L working volume fermentor containing 2×YT broth andboth Kn and Ap antibiotics. The fermentor (Bioflo 3000, New BrunswickScientific) was operated at 37±1° C., 0.2-0.4 VVM air sparge, 250 rpm inRushton impellers. The pH was not controlled in either the flask seedculture or the fermentor. During fermentation, the pH ranged 6.5 to 7.3in the fermentor. IPTG (1.0 M stock, prepared in sterile water) wasadded to the fermentor when the culture reached mid-log of growth (˜0.7O.D. 600 units). Cells were induced for 2-4 hours then harvested bycentrifugation using either a 28RS Heracus (Sepatech) or RC5C superspeedcentrifuge (Sorvall Instruments). Cell paste was stored at −20 C untilprocessed.

[0229] Purification

[0230] Chemicals and Materials

[0231] Imidazole, guanidine hydrochloride, Tris (hydroxymethyl), andEDTA (ethylene-diamine tetraacetic acid) biotechnology grade or betterwere all obtained from Ameresco Chemical, Solon, Ohio. Triton X-100(t-Octylphenoxypolyethoxy-ethanol), sodium phosphate, monobasic, andUrea were reagent grade or better and obtained from Sigma ChemicalCompany, St. Louis, Mo. Glacial acetic acid and hydrochloric acid wereobtained from Mallincrodt Baker Inc., Phillipsburg, N.J. Methanol wasobtained from Fisher Scientific, Fairlawn, N.J. Pefabloc®SC(4-(2-Aminoethyl)-benzenesulfonylfuoride), Complete protease inhibitorcocktail tablets, and PMSF (phenylmethyl-sulfonylfluoride) were obtainedfrom Roche Diagnostics Corporation, Indianapolis, Ind. Bestatin,Pepstatin A, and E-64 protease inhibitor were obtained from Calbiochem,LaJolla, Calif. Dulbecco's Phosphate Buffered Saline(1×PBS) was obtainedfrom Quality Biological, Inc., Gaithersburg, Md. Dulbecco's PhosphateBuffered Saline (10×PBS) was obtained from BioWhittaker, Walkersville,Md. Penta-His Antibody, BSA free was obtained from QiaGen, Valencia,Calif. Peroxidase-conjugated AffiniPure Goat Anti-mouse IgG was obtainedfrom Jackson Immuno Research, West Grove, Penn. AEC single solution wasobtained from Zymed, South San Francisco, Calif. All other chemicalswere reagent grade or better.

[0232] Ni-NTA Superflow resin was obtained from QiaGen Inc., Valencia,Calif. Precast Tris-Glycine 4-20% and 10-20% polyacrylamide gels, allrunning buffers and solutions, SeeBlue Pre-Stained Standards, MultiMarkMulti-Colored Standards and PVDF transfer membranes were obtained fromNovex, San Diego, Calif. SDS-PAGE Silver Stain kits were obtained fromDaiichi Pure Chemicals Company Limited, Tokyo, Japan. Coomassie StainSolution was obtained from Bio-Rad Laboratories, Hercules, Calif.Acrodisc® PF 0.2 m syringe filters were obtained from Pall GelmanSciences, Ann Arbor, Mich. GD/X 25 mm disposable syringe filters wereobtained from Whatman Inc., Clifton, N.J. Dialysis tubing 8,000 MWCO wasobtained from BioDesign Inc. Od New York, Carmal N.Y. BCA Protein AssayReagents and Snake Skin dialysis tubing 3,500 MWCO were obtained fromPierce Chemical Co., Rockford, Ill.

[0233] Extraction Protocol

[0234] Cell paste was thawed at room temperature for 30 to 60 minutes.Five to six grams of material was weighed out into a 50 ml disposablecentrifuge tube. To this five mls/gram of Guanidine hydrochloride(Gu-HCl) buffer was added (6 M Guanidine hydrochloride, 100 mM Sodiumphosphate, monobasic, 10 mM Tris and 0.05% Triton X-100, pH 8.0). Cellpaste was resuspended using a PRO300D proscientific homogenizer, at ¾power for one minute. The extraction mixture was then placed at roomtemperature with gentle agitation for 60 to 90 minutes. After 60 to 90minutes the extraction mixture was centrifuged at 15,800×g for 15minutes (Sorvall RC5C centrifuge, 11,500 rpm). The supernatant (S1) wasdecanted and saved for additional purification. The pellet (P1) wassaved for analysis.

[0235] Binding of BASB034 to Nickel-NTA Resin

[0236] To the S1 three to four mls of Ni-NTA resin is added. This isthen placed at room temperature with gentle agitation for one hour.After one hour the S1/Ni-NTA is packed into an XK16 Pharmacia column.The column is then washed with 1 M Gu-HCl buffer (1 M Guanidinehydrochloride, 100 mM Sodium phosphate, monobasic, 10 mM Tris and 0.05%Triton X-100, pH 8.0). This is then followed by a wash with phosphatebuffer (100 mM Sodium phosphate, monobasic, 10 mM Tris and 0.05% TritonX-100, pH 6.3). The protein is then eluted from the column with a 250 mMimidazole buffer (250 mM imidazole, 100 mM Sodium phosphate, monobasic,10 mM Tris and 0.05% Triton X-100, pH 5.9).

[0237] Final Formulation

[0238] BASB034 was formulated by dialysis overnight against, threechanges of 0.1% Triton X-100 and lx PBS, pH 7.4, to remove residualGu-HCl and imidazole. The purified protein was characterized and used toproduced antibodies as described below.

[0239] Biochemical Characterizations

[0240] SDS-PAGE and Western Blot Analysis

[0241] The recombinant purified protein was resolved on 4-20%polyacrylamide gels and electrophoretically transferred to PVDFmembranes at 100 V for 1 hour as previously described (Thebaine et al.1979, Proc. Natl. Acad. Sci. USA 76:4350-4354). The PVDF membranes werethen pretreated with 25 ml of Dulbeeco's phosphate buffered salinecontaining 5% non-fat dry milk. All subsequent incubations were carriedout using this pretreatment buffer.

[0242] PVDF membranes were incubated with 25 ml of a 1:500 dilution ofpreimmune serum or rabbit anti-His immune serum for 1 hour at roomtemperature. PVDF membranes were then washed twice with wash buffer (20mM Tris buffer, pH 7.5, containing 150 mM sodium chloride and 0.05%Tween-20). PVDF membranes were incubated with 25 ml of a 1:5000 dilutionof peroxidase-labeled goat anti-rabbit IgG (Jackson ImmunoResearchLaboratories, West Grove, Pa.) for 30 minutes at room temperature. PVDFmembranes were then washed 4 times with wash buffer, and were developedwith 3-amino-9-ethylcarbazole and urea peroxide as supplied by Zymed(San Francisco, Calif.) for 10 minutes each.

[0243] The results of an SDS-PAGE (FIG. 4) show a protein about 60 kDathat is reactive to an anti-RGS(His) antibody by western blots (FIG. 5)of the SDS-PAGE.

[0244] Protein Sequencing

[0245] Amino terminal amino acid sequencing of the purified protein wasperformed to confirm the production of the correct recombinant proteinusing well defined chemical protocols on Hewlett-Packard model G10000Asequencer with a model 1090 LC and a Hewlett-Packard model 241 sequencerwith a model 1100 LC.

Example 5 Production of Antisera to Recombinant BASB034

[0246] Polyvalent antisera directed against the BASB034 protein weregenerated by vaccinating two rabbits with the purified recombinantBASB034 protein. Each animal is given a total of three immunizationsintramuscullarly (i.m.) of about 20 μg BASB034 protein per injection(beginning with complete Freund's adjuvant and followed with incompleteFreund's adjuvant) at approximately 21 day intervals. Animals were bledprior to the first immunization (“pre-bleed”) and on days 35 and 57.

[0247] Anti-BASB034 protein titres were measured by an ELISA usingpurified recombinant BASB034 protein (0.5 μg/well). The titre is definedas the highest dilution equal or greater than 0.1 as calculated with thefollowing equation: average OD of two test samples of antisera—theaverage OD of two test samples of buffer. The titres after threeimmunizations were around 1000000.

[0248] The antisera were used as the first antibody to identify theprotein in a western blot as described in example 4 above. Thewestern-blot shows the presence of anti-BASB034 antibody in the sera ofimmunized animals (FIG. 6).

Example 6 Analysis of the Non-Coding Flanking Regions of the BASB034Gene and its Exploitation for Modulated BASB034 Gene Expression.

[0249] The non-coding flanking regions of the BASB034 gene containregulatory elements important in the expression of the gene. Thisregulation takes place both at the transcriptional and translationallevel. The sequence of these regions, either upstream or downstream ofthe open reading frame of the gene, can be obtained by DNA sequencing.This sequence information allows the determination of potentialregulatory motifs such as the different promoter elements, terminatorsequences, inducible sequence elements; repressors, elements responsiblefor phase variation, the shine-dalgarno sequence, regions with potentialsecondary structure involved in regulation, as well as other types ofregulatory motifs or sequences.

[0250] This sequence information allows the modulation of the naturalexpression of gene BASB034. The upregulation of the gene expression maybe accomplished by altering the promoter, the shine-dalgarno sequence,potential repressor or operator elements, or any other elementsinvolved. Likewise, downregulation of expression can be achieved bysimilar types of modifications. Alternatively, by changing phasevariation sequences, the expression of the gene can be put under phasevariation control, or may be uncoupled from this regulation. In anotherapproach, the expression of the gene can be put under the control of oneor more inducible elements allowing regulated expression. Examples ofsuch regulation include, but are not limited to, induction bytemperature shift, addition of inductor substrates like selectedcarbohydrates or their derivatives, trace elements, vitamins,co-factors, metal ions, etc.

[0251] Such modifications as described above can be introduced byseveral different means. The modification of sequences involved in geneexpression can be done in vivo by random mutagenesis followed byselection for the desired phenotype. Another approach consists inisolating the region of interest and modifying it by random mutagenesis,or site-directed mutagenesis, insertion or deletion mutagenesis. Themodified region can then be reintroduced into the bacterial genome byhomologous recombination, and the effect on gene expression can beassessed. In another approach, the sequence knowledge of the region ofinterest can be used to replace or delete all or part of the naturalregulatory sequences. In this case, the regulatory region targeted isisolated and modified so as to contain the regulatory elements fromanother gene, a combination of regulatory elements from different genes,a synthetic regulatory region, or any other regulatory region, or todelete selected parts of the wild-type regulatory sequences. Thesemodified sequences can then be reintroduced into the bacterium viahomologous recombination into the genome. A non-exhaustive list ofpreferred promoters that could be used for up-regulation of geneexpression includes the promoter porA, porB, lbpB, tbpB, p110, lst,hpuAB from N. meningitidis or N. gonorroheae.

[0252] In one example, the expression of the gene can be modulated byexchanging its promoter with a stronger promoter (through isolating theupstream sequence of the gene, in vitro modification of this sequence,and reintroduction into the genome by homologous recombination).Upregulated expression can be obtained in both the bacterium as well asin the outer membrane vesicles shed (or made) from the bacterium.

[0253] In other examples, the described approaches can be used togenerate recombinant bacterial strains with improved characteristics forvaccine applications. These can be, but are not limited to, attenuatedstrains, strains with increased expression of selected antigens, strainswith knock-outs (or decreased expression) of genes interfering with theimmune response, strains with modulated expression of immounodominantproteins, strains with modulated shedding of outer-membrane vesicles.

[0254] A region directly upstream of the BASB034 gene is given in thesequence of SEQ ID NO:13. This sequence is a further aspect of theinvention.

[0255] Deposited Materials

[0256] A deposit containing a Moraxella catarrhalis Catlin strain hasbeen deposited with the American Type Culture Collection (herein “ATCC”)on Jun. 21, 1997 and assigned deposit number 43617. The deposit wasdescribed as Branhamella catarrhalis (Frosch and Kolle) and is afreeze-dried, 1.5-2.9 kb insert library constructed from M. catarrhalisisolate obtained from a transtracheal aspirate of a coal miner withchronic bronchitits. The deposit is described in Antimicrob. AgentsChemother. 21: 506-508 (1982).

[0257] The Moraxella catarrhalis strain deposit is referred to herein as“the deposited strain” or as “the DNA of the deposited strain.”

[0258] The deposited strain contains a full length BASB034 gene.

[0259] A deposit of the vector pMC-PLA1 consisting of Moraxellacatarrhalis DNA inserted in pQE30 has been deposited with the AmericanType Culture Collection (ATCC) on Feb. 12, 1999 and assigned depositnumber 207099.

[0260] The sequence of the polynucleotides contained in the depositedstrain, or in the deposited clone, as well as the amino acid sequence ofany polypeptide encoded thereby, are controlling in the event of anyconflict with any description of sequences herein.

[0261] The deposits of the deposited strain/clone have been made underthe terms of the Budapest Treaty on the International Recognition of theDeposit of Micro-organisms for Purposes of Patent Procedure. Thedeposited strains will be irrevocably and without restriction orcondition released to the public upon the issuance of a patent. Thedeposited strains are provided merely as convenience to those of skillin the art and are not an admission that a deposit is required forenablement, such as that required under 35 U.S.C. §112.

1 13 1 1329 DNA Moraxella catarrhalis 1 atgaaagttt cactgtctac attgactttatctattttgt catgttttgc tatcctagcc 60 attcagcaag cacaagctgt accaaatcctgtggcatttg ttgacgaagt acgcagtgaa 120 aatgatcttg ggcaagacaa tgaattacccattgatgtcc aaagtgcgac acaatcagcg 180 tctactgata cggctaatcc tttagacgaacatgaaccag agctttatac gacagcttta 240 gaaaataaaa ccatgctgat taactgctcagcacttaatc aagatatcat gcgtttggcg 300 tgctatgaca ctttggtgca tggtgagacgccagcggtaa ttaaaaccaa gcgttccatt 360 cgccttgatg aaacaatttg gcagaccatcaaaggcaaac cccaggttat ctatcaagaa 420 acgacagatc cgattttttt aatgggtaatgaaaaaggca tgctgaccaa aaaagatgcc 480 aaacagcttg aatatgcagc caaacagtttacaccactga gcttatcatt tgatttagac 540 cgaaataata caccactttg gtcatcacgaccacacaatc cgatgtatgt attgcccata 600 tttatgcacg gtaagcctaa tcgaagcccaaatacgccca gtcatgaagc aaaacaattt 660 accccaaatg aatttcgtgc tcccgagctaaaatttcagg tttctgttaa ggttaaagct 720 gctgaggatt tatgggggac ggattcagatttatggtttg gatatacaca gcaatcgcac 780 tggcagattt ttaatggaaa aaactctcgtccttttagag tacatgacta ccagccagag 840 attttcttaa ctcaacctgt atactcagacttaccatggg atggcaaagt ccgcatgatt 900 ggcatgggtg cggtacatca ttccaatggtgaaagtgcca aactgtctcg ctcatggaat 960 cgtgcttatt tgatggcagg catggaatggaaaaacctga ctgtcatgcc acgcatttgg 1020 gggcgtatct ttaaagaggg tagtggcagccagccagatg ataatcctga tatcttggac 1080 tattatggtt atggtgatgt gcgttttttatatcaactag aaaataaaag taatatttca 1140 ggtacggtac gctataatcc acgctcaggcaaaggtgcgt tgcaacttga ctatgtctat 1200 ccgcttggta agggaattag tggctattttcaaatatttc aaggctatgg gcagtctttg 1260 attgattata atcatgaggc gacaagctttggcgtcggac ttatgcttaa cgactggatg 1320 ggtctataa 1329 2 442 PRT Moraxellacatarrhalis 2 Met Lys Val Ser Leu Ser Thr Leu Thr Leu Ser Ile Leu SerCys Phe 1 5 10 15 Ala Ile Leu Ala Ile Gln Gln Ala Gln Ala Val Pro AsnPro Val Ala 20 25 30 Phe Val Asp Glu Val Arg Ser Glu Asn Asp Leu Gly GlnAsp Asn Glu 35 40 45 Leu Pro Ile Asp Val Gln Ser Ala Thr Gln Ser Ala SerThr Asp Thr 50 55 60 Ala Asn Pro Leu Asp Glu His Glu Pro Glu Leu Tyr ThrThr Ala Leu 65 70 75 80 Glu Asn Lys Thr Met Leu Ile Asn Cys Ser Ala LeuAsn Gln Asp Ile 85 90 95 Met Arg Leu Ala Cys Tyr Asp Thr Leu Val His GlyGlu Thr Pro Ala 100 105 110 Val Ile Lys Thr Lys Arg Ser Ile Arg Leu AspGlu Thr Ile Trp Gln 115 120 125 Thr Ile Lys Gly Lys Pro Gln Val Ile TyrGln Glu Thr Thr Asp Pro 130 135 140 Ile Phe Leu Met Gly Asn Glu Lys GlyMet Leu Thr Lys Lys Asp Ala 145 150 155 160 Lys Gln Leu Glu Tyr Ala AlaLys Gln Phe Thr Pro Leu Ser Leu Ser 165 170 175 Phe Asp Leu Asp Arg AsnAsn Thr Pro Leu Trp Ser Ser Arg Pro His 180 185 190 Asn Pro Met Tyr ValLeu Pro Ile Phe Met His Gly Lys Pro Asn Arg 195 200 205 Ser Pro Asn ThrPro Ser His Glu Ala Lys Gln Phe Thr Pro Asn Glu 210 215 220 Phe Arg AlaPro Glu Leu Lys Phe Gln Val Ser Val Lys Val Lys Ala 225 230 235 240 AlaGlu Asp Leu Trp Gly Thr Asp Ser Asp Leu Trp Phe Gly Tyr Thr 245 250 255Gln Gln Ser His Trp Gln Ile Phe Asn Gly Lys Asn Ser Arg Pro Phe 260 265270 Arg Val His Asp Tyr Gln Pro Glu Ile Phe Leu Thr Gln Pro Val Tyr 275280 285 Ser Asp Leu Pro Trp Asp Gly Lys Val Arg Met Ile Gly Met Gly Ala290 295 300 Val His His Ser Asn Gly Glu Ser Ala Lys Leu Ser Arg Ser TrpAsn 305 310 315 320 Arg Ala Tyr Leu Met Ala Gly Met Glu Trp Lys Asn LeuThr Val Met 325 330 335 Pro Arg Ile Trp Gly Arg Ile Phe Lys Glu Gly SerGly Ser Gln Pro 340 345 350 Asp Asp Asn Pro Asp Ile Leu Asp Tyr Tyr GlyTyr Gly Asp Val Arg 355 360 365 Phe Leu Tyr Gln Leu Glu Asn Lys Ser AsnIle Ser Gly Thr Val Arg 370 375 380 Tyr Asn Pro Arg Ser Gly Lys Gly AlaLeu Gln Leu Asp Tyr Val Tyr 385 390 395 400 Pro Leu Gly Lys Gly Ile SerGly Tyr Phe Gln Ile Phe Gln Gly Tyr 405 410 415 Gly Gln Ser Leu Ile AspTyr Asn His Glu Ala Thr Ser Phe Gly Val 420 425 430 Gly Leu Met Leu AsnAsp Trp Met Gly Leu 435 440 3 1329 DNA Moraxella catarrhalis 3atgaaagttt cactgtctac attgacttta tctattttgc catgttttgc tatcctagcc 60attcagcaag cacaagctgt accaaatcct gtggcatttg ttgacgaagt acgcagtaaa 120aatgatcttg ggcaagacaa tgaattactc attggtgtac aaagtgcgac acaatcagcg 180tctactgata cggctaatcc tttagacgaa catgaaccag agctttatac gacagcttta 240gaaaataaaa ccatgctgat taactgctca gcacttaatc aagatatcat gcgtttggcg 300tgctatgaca ctttggtgca tggtgagacg ccagcggtaa ttaaaaccaa gcgttccatt 360cgccttgatg aaacaatttg gcagaccatc aaaggcaaac cccaggttgt ctatcaagaa 420acgacagatc cgattttttt aatgggtaat gaaaaaggca tgctgaccaa aaaagatgcc 480aaacagcttg aatatgcagc caaacagttt acaccactga gcttatcatt tgatttagac 540cgaaataata caccgctttg gtcatcacga ccacacaatc cgatgtatgt attgcccata 600tttatgcacg gtaagcctaa tcgaagccca aatacgccca gtcatgaagc aagacaattt 660accccaaatg aatttcgtgc ccctgaatta aaatttcaag tttctgttaa ggttaaagct 720gctgaggatt tatgggggac ggattcagat ttatggtttg ggtatacaca gcaatcgcac 780tggcagattt ttaatggaaa aaactctcgt ccttttagag tacatgatta ccagccagag 840attttcttaa ctcaacctgt gtactcagac ttaccatggg atggcaaagt ccgcatgatt 900ggcatgggtg cggtacatca ttccaatggt gaaagtgcca aactgtctcg ctcatggaat 960cgtgcttatt tgatggcagg catggaatgg aaaaacctga ctgtcatgcc acgcatttgg 1020gggcgtatct ttaaagaggg tagtggcagc cagccagatg acaatcctga tatcttggac 1080tattatggtt atggtgatgt gcgtttttta tatcaactag aaaataaaag taatatttca 1140ggtacggtac gctataatcc acgctcaggc aaaggtgcgt tgcaacttga ctatgtctat 1200ccgcttggta agggaattag tggctatttt caaatatttc aaggctatgg gcagtctttg 1260attgattata atcatgaggc gacaagcttt ggcgtcggac ttatgcttaa cgactggatg 1320ggtctataa 1329 4 442 PRT Moraxella catarrhalis 4 Met Lys Val Ser Leu SerThr Leu Thr Leu Ser Ile Leu Pro Cys Phe 1 5 10 15 Ala Ile Leu Ala IleGln Gln Ala Gln Ala Val Pro Asn Pro Val Ala 20 25 30 Phe Val Asp Glu ValArg Ser Lys Asn Asp Leu Gly Gln Asp Asn Glu 35 40 45 Leu Leu Ile Gly ValGln Ser Ala Thr Gln Ser Ala Ser Thr Asp Thr 50 55 60 Ala Asn Pro Leu AspGlu His Glu Pro Glu Leu Tyr Thr Thr Ala Leu 65 70 75 80 Glu Asn Lys ThrMet Leu Ile Asn Cys Ser Ala Leu Asn Gln Asp Ile 85 90 95 Met Arg Leu AlaCys Tyr Asp Thr Leu Val His Gly Glu Thr Pro Ala 100 105 110 Val Ile LysThr Lys Arg Ser Ile Arg Leu Asp Glu Thr Ile Trp Gln 115 120 125 Thr IleLys Gly Lys Pro Gln Val Val Tyr Gln Glu Thr Thr Asp Pro 130 135 140 IlePhe Leu Met Gly Asn Glu Lys Gly Met Leu Thr Lys Lys Asp Ala 145 150 155160 Lys Gln Leu Glu Tyr Ala Ala Lys Gln Phe Thr Pro Leu Ser Leu Ser 165170 175 Phe Asp Leu Asp Arg Asn Asn Thr Pro Leu Trp Ser Ser Arg Pro His180 185 190 Asn Pro Met Tyr Val Leu Pro Ile Phe Met His Gly Lys Pro AsnArg 195 200 205 Ser Pro Asn Thr Pro Ser His Glu Ala Arg Gln Phe Thr ProAsn Glu 210 215 220 Phe Arg Ala Pro Glu Leu Lys Phe Gln Val Ser Val LysVal Lys Ala 225 230 235 240 Ala Glu Asp Leu Trp Gly Thr Asp Ser Asp LeuTrp Phe Gly Tyr Thr 245 250 255 Gln Gln Ser His Trp Gln Ile Phe Asn GlyLys Asn Ser Arg Pro Phe 260 265 270 Arg Val His Asp Tyr Gln Pro Glu IlePhe Leu Thr Gln Pro Val Tyr 275 280 285 Ser Asp Leu Pro Trp Asp Gly LysVal Arg Met Ile Gly Met Gly Ala 290 295 300 Val His His Ser Asn Gly GluSer Ala Lys Leu Ser Arg Ser Trp Asn 305 310 315 320 Arg Ala Tyr Leu MetAla Gly Met Glu Trp Lys Asn Leu Thr Val Met 325 330 335 Pro Arg Ile TrpGly Arg Ile Phe Lys Glu Gly Ser Gly Ser Gln Pro 340 345 350 Asp Asp AsnPro Asp Ile Leu Asp Tyr Tyr Gly Tyr Gly Asp Val Arg 355 360 365 Phe LeuTyr Gln Leu Glu Asn Lys Ser Asn Ile Ser Gly Thr Val Arg 370 375 380 TyrAsn Pro Arg Ser Gly Lys Gly Ala Leu Gln Leu Asp Tyr Val Tyr 385 390 395400 Pro Leu Gly Lys Gly Ile Ser Gly Tyr Phe Gln Ile Phe Gln Gly Tyr 405410 415 Gly Gln Ser Leu Ile Asp Tyr Asn His Glu Ala Thr Ser Phe Gly Val420 425 430 Gly Leu Met Leu Asn Asp Trp Met Gly Leu 435 440 5 1329 DNAMoraxella catarrhalis 5 atgaaagttt cactgtctac attgacttta tctattttgtcatgttttgc tatcctagcc 60 attcagcaag caaaagctgt accaaatcct gtggcatttgttgacgaagt acgcagtgaa 120 aatgatcttg ggcaagacaa tgaattaccc attgatgtccaaagtgcgac acaatcagcg 180 tctactgata cggctaatcc tttagacgaa catgaaccagagctttatac gacagcttta 240 gaaaataaaa ccatgctgat taactgctca gcacttaatcaagatatcat gcgtttggcg 300 tgctatgaca ctttggtgca tggtgagacg ccagcggtaattaaaaccaa gcgttccatt 360 cgccttgatg aaacaatttg gcagaccatc aaaggcaaaccccaggttgt ctatcaagaa 420 acgacagatc cgattttttt aatgggtaat gaaaaaggcatgctgaccaa aaaagatgcc 480 aaacagcttg aatatgcagc caaacagttt acaccactgagcttatcatt tgatttagac 540 cgaaataata caccactttg gtcatcacga ccacacaatccgatgtatgt attgcccata 600 tttatgcacg gtaagcctaa tcgaagccca aatacgcccagtcatgaagc aagacaattt 660 accccaaatg aatttcgtgc ccctgaatta aaatttcaagtttctgttaa ggttaaagct 720 gctgaggatt tatgggggac ggattcagat ttatggtttggatatacaca gcaatcgcac 780 tggcagattt ttaatggaaa aaactctcgt ccttttagagtacatgatta ccagccagag 840 attttcttaa ctcaacctgt atactcagac ttaccatgggatggcaaagt ccgcatgatt 900 ggcatgggtg cggtacatca ttccaatggt gaaagtgccaaactgtctcg ctcatggaat 960 cgtgcttatt tgatggcagg catggaatgg aaaaacctgactgtcatgcc acgcatttgg 1020 gggcgtatct ttaaagaggg tagtggcagc cagccagatgacaatcctga tatcttggac 1080 tattatggtt atggtgatgt gcgtttttta tatcaactagaaaataaaag taatatttca 1140 ggtacggtac gctataatcc acgctcaggc aaaggtgcgttgcaacttga ctatgtctat 1200 ccgcttggta agggaattag tggctatttt caaatatttcaaggctatgg gcagtctttg 1260 attgattata atcatgaggc gacaagcttt ggcgtcggacttatgcttaa cgactggatg 1320 ggtctataa 1329 6 442 PRT Moraxellacatarrhalis 6 Met Lys Val Ser Leu Ser Thr Leu Thr Leu Ser Ile Leu SerCys Phe 1 5 10 15 Ala Ile Leu Ala Ile Gln Gln Ala Lys Ala Val Pro AsnPro Val Ala 20 25 30 Phe Val Asp Glu Val Arg Ser Glu Asn Asp Leu Gly GlnAsp Asn Glu 35 40 45 Leu Pro Ile Asp Val Gln Ser Ala Thr Gln Ser Ala SerThr Asp Thr 50 55 60 Ala Asn Pro Leu Asp Glu His Glu Pro Glu Leu Tyr ThrThr Ala Leu 65 70 75 80 Glu Asn Lys Thr Met Leu Ile Asn Cys Ser Ala LeuAsn Gln Asp Ile 85 90 95 Met Arg Leu Ala Cys Tyr Asp Thr Leu Val His GlyGlu Thr Pro Ala 100 105 110 Val Ile Lys Thr Lys Arg Ser Ile Arg Leu AspGlu Thr Ile Trp Gln 115 120 125 Thr Ile Lys Gly Lys Pro Gln Val Val TyrGln Glu Thr Thr Asp Pro 130 135 140 Ile Phe Leu Met Gly Asn Glu Lys GlyMet Leu Thr Lys Lys Asp Ala 145 150 155 160 Lys Gln Leu Glu Tyr Ala AlaLys Gln Phe Thr Pro Leu Ser Leu Ser 165 170 175 Phe Asp Leu Asp Arg AsnAsn Thr Pro Leu Trp Ser Ser Arg Pro His 180 185 190 Asn Pro Met Tyr ValLeu Pro Ile Phe Met His Gly Lys Pro Asn Arg 195 200 205 Ser Pro Asn ThrPro Ser His Glu Ala Arg Gln Phe Thr Pro Asn Glu 210 215 220 Phe Arg AlaPro Glu Leu Lys Phe Gln Val Ser Val Lys Val Lys Ala 225 230 235 240 AlaGlu Asp Leu Trp Gly Thr Asp Ser Asp Leu Trp Phe Gly Tyr Thr 245 250 255Gln Gln Ser His Trp Gln Ile Phe Asn Gly Lys Asn Ser Arg Pro Phe 260 265270 Arg Val His Asp Tyr Gln Pro Glu Ile Phe Leu Thr Gln Pro Val Tyr 275280 285 Ser Asp Leu Pro Trp Asp Gly Lys Val Arg Met Ile Gly Met Gly Ala290 295 300 Val His His Ser Asn Gly Glu Ser Ala Lys Leu Ser Arg Ser TrpAsn 305 310 315 320 Arg Ala Tyr Leu Met Ala Gly Met Glu Trp Lys Asn LeuThr Val Met 325 330 335 Pro Arg Ile Trp Gly Arg Ile Phe Lys Glu Gly SerGly Ser Gln Pro 340 345 350 Asp Asp Asn Pro Asp Ile Leu Asp Tyr Tyr GlyTyr Gly Asp Val Arg 355 360 365 Phe Leu Tyr Gln Leu Glu Asn Lys Ser AsnIle Ser Gly Thr Val Arg 370 375 380 Tyr Asn Pro Arg Ser Gly Lys Gly AlaLeu Gln Leu Asp Tyr Val Tyr 385 390 395 400 Pro Leu Gly Lys Gly Ile SerGly Tyr Phe Gln Ile Phe Gln Gly Tyr 405 410 415 Gly Gln Ser Leu Ile AspTyr Asn His Glu Ala Thr Ser Phe Gly Val 420 425 430 Gly Leu Met Leu AsnAsp Trp Met Gly Leu 435 440 7 1329 DNA Moraxella catarrhalis 7atgaaagttt cactgtctac attgacttta tctattttgc catgttttgc catcctagcc 60attcagcaag cacaagctgt accaaatcct gtggcatttg ttgacgaagt acgcagtgaa 120aatgatcttg ggcaagacaa tgaattaccc attgatgtcc aaagtgcgac acaatcggcg 180tctactgata cggctaatcc tttagacgaa catgaaccag agctttatac gacagcttta 240gaaaataaaa ccatgctgat taactgctca gcacttaatc aagatatcat gcgtttggcg 300tgctatgaca ctttggtgca tggtgagacg ccagcggtaa ttaaaaccaa gcgttccatt 360cgccttgatg aaacaatttg gcagaccatc aaaggcaaac cccaggttgt ctatcaagaa 420acgacagatc cgattttttt aatgggtaat gaaaaaggca tgctgaccaa aaaagatgcc 480aaacagcttg aatatgcagc caaacagttt acaccactga gcttatcatt tgatttagac 540cgaaataata caccactttg gtcatcacga ccacacaatc cgatgtatgt attgcccata 600tttatgcacg gtaagcctaa tcgaagccca aatacgccca gtcatgaagc aaaacaattt 660accccaaatg aatttcgtgc tcccgagcta aaatttcagg tttctgttaa ggttaaagct 720gctgaggatt tatgggggac ggattcagat ttatggtttg gatatacaca gcaatcgcac 780tggcagattt ttaatggaaa aaactctcgt ccttttagag tacatgacta ccagccagag 840attttcttaa ctcaacctgt atactcagac ttaccatggg atggcaaagt ccgcatgatt 900ggcatgggtg cggtacatca ttccaatggt gaaagtgcca aactgtctcg ctcatggaat 960cgtgcttatt tgatggcagg catggaatgg aaaaacctga ctgtcatgcc acgcatttgg 1020gggcgtatct ttaaagaggg tagtggcagc cagccagatg ataatcctga tatcttggac 1080tattatggtt atggtgatgt gcgtttttta tatcaactag aaaataaaag taatatttca 1140ggtacggtac gctataatcc acgctcaggc aaaggtgcgt tgcaacttga ctatgtctat 1200ccgcttggta agggaattag tggctatttt caaatatttc aaggctatgg gcagtctttg 1260attgattata atcatgaggc gacaagcttt ggcgtcggac ttatgcttaa cgactggatg 1320ggtctataa 1329 8 442 PRT Moraxella catarrhalis 8 Met Lys Val Ser Leu SerThr Leu Thr Leu Ser Ile Leu Pro Cys Phe 1 5 10 15 Ala Ile Leu Ala IleGln Gln Ala Gln Ala Val Pro Asn Pro Val Ala 20 25 30 Phe Val Asp Glu ValArg Ser Glu Asn Asp Leu Gly Gln Asp Asn Glu 35 40 45 Leu Pro Ile Asp ValGln Ser Ala Thr Gln Ser Ala Ser Thr Asp Thr 50 55 60 Ala Asn Pro Leu AspGlu His Glu Pro Glu Leu Tyr Thr Thr Ala Leu 65 70 75 80 Glu Asn Lys ThrMet Leu Ile Asn Cys Ser Ala Leu Asn Gln Asp Ile 85 90 95 Met Arg Leu AlaCys Tyr Asp Thr Leu Val His Gly Glu Thr Pro Ala 100 105 110 Val Ile LysThr Lys Arg Ser Ile Arg Leu Asp Glu Thr Ile Trp Gln 115 120 125 Thr IleLys Gly Lys Pro Gln Val Val Tyr Gln Glu Thr Thr Asp Pro 130 135 140 IlePhe Leu Met Gly Asn Glu Lys Gly Met Leu Thr Lys Lys Asp Ala 145 150 155160 Lys Gln Leu Glu Tyr Ala Ala Lys Gln Phe Thr Pro Leu Ser Leu Ser 165170 175 Phe Asp Leu Asp Arg Asn Asn Thr Pro Leu Trp Ser Ser Arg Pro His180 185 190 Asn Pro Met Tyr Val Leu Pro Ile Phe Met His Gly Lys Pro AsnArg 195 200 205 Ser Pro Asn Thr Pro Ser His Glu Ala Lys Gln Phe Thr ProAsn Glu 210 215 220 Phe Arg Ala Pro Glu Leu Lys Phe Gln Val Ser Val LysVal Lys Ala 225 230 235 240 Ala Glu Asp Leu Trp Gly Thr Asp Ser Asp LeuTrp Phe Gly Tyr Thr 245 250 255 Gln Gln Ser His Trp Gln Ile Phe Asn GlyLys Asn Ser Arg Pro Phe 260 265 270 Arg Val His Asp Tyr Gln Pro Glu IlePhe Leu Thr Gln Pro Val Tyr 275 280 285 Ser Asp Leu Pro Trp Asp Gly LysVal Arg Met Ile Gly Met Gly Ala 290 295 300 Val His His Ser Asn Gly GluSer Ala Lys Leu Ser Arg Ser Trp Asn 305 310 315 320 Arg Ala Tyr Leu MetAla Gly Met Glu Trp Lys Asn Leu Thr Val Met 325 330 335 Pro Arg Ile TrpGly Arg Ile Phe Lys Glu Gly Ser Gly Ser Gln Pro 340 345 350 Asp Asp AsnPro Asp Ile Leu Asp Tyr Tyr Gly Tyr Gly Asp Val Arg 355 360 365 Phe LeuTyr Gln Leu Glu Asn Lys Ser Asn Ile Ser Gly Thr Val Arg 370 375 380 TyrAsn Pro Arg Ser Gly Lys Gly Ala Leu Gln Leu Asp Tyr Val Tyr 385 390 395400 Pro Leu Gly Lys Gly Ile Ser Gly Tyr Phe Gln Ile Phe Gln Gly Tyr 405410 415 Gly Gln Ser Leu Ile Asp Tyr Asn His Glu Ala Thr Ser Phe Gly Val420 425 430 Gly Leu Met Leu Asn Asp Trp Met Gly Leu 435 440 9 22 DNAArtificial Sequence Primer sequence 9 gatttaagag tatgttatga tg 22 10 21DNA Artificial Sequence Primer sequence 10 gtatgggttg atcaaataca g 21 1158 DNA Artificial Sequence Oligonucleotide 11 aagggcccaa ttacgcagaggggatcccaa gctgtaccaa atcctgtggc atttgttg 58 12 60 DNA ArtificialSequence Oligonucleotide 12 aagggcccaa ttacgcagag ggtcgactta ttatagacccatccagtcgt taagcataag 60 13 1000 DNA Moraxella catarrhalis 13 acttggcgaaaataccattt atatcgattg tgatgttata caggcagatg gcggtacacg 60 cacagccagtatcagtggtg ctgcggtggc acttattgat gctttagaac acttgcagcg 120 tcgtaaaaagcttacccaag atccgctttt gggcttggtg gcagcggttt ctgtgggtgt 180 taatcaaggccgtgtattgc ttgatttgga ttatgctgaa gattcaactt gtgataccga 240 tttaaatgtggtcatgacgc aggcaggtgg gtttattgag attcaaggca cagcagaaga 300 aaagccatttactcgtgctg aagctaatgc gatgcttgat ttggcagagc tgggaattgg 360 gcagattatcgaagcccaaa agcaagtatt aggctggtga tatgctaatc gttgaagata 420 atggcgtgatcatcacatta aatggacaag taaaagaccc attattttgg tggtcgatga 480 tattgctgctgctgggtgtc ttggtggcaa tcatttgttt gattgcaccc gttttttatg 540 caatcggtgcgttggcttta tttgcagttg tggtatttgt gtttaatatt caaaggcaaa 600 aagccaaaacttgtcatatg ttttcacaag gtcgcttgaa gattacgtcc aaacgctttg 660 agattcataacaaatcacta accttatcag catcggcaac aatatctgct aaagataaca 720 aaatgacaattgttgatcgg ggcattgaat atcattttac aggttttgct gatgaccgtg 780 aaattaatatagccaaacag gtacttttgg gaaagtcaat caaaaccaat gcggtggcgg 840 taacattggctaagtagttg ttgtgataca gacaggttgg atggtcttta actccaccca 900 cctaactttttctttgtttg gatttaagag tatgttatga tgggcaggat tttattttaa 960 gtcatcatttaatgcaatca gttgtccaga gtagccgttc 1000

What is claimed is:
 1. An isolated polynucleotide comprising a firstpolynucleotide sequence or the full complement of the firstpolynucleotide sequence, wherein the first polynucleotide sequenceencodes a polypeptide selected from the group consisting of SEQ ID NO:2,4, 6 or
 8. 2. The isolated polynucleotide of claim 1, wherein theisolated polynucleotide comprises the first polynucleotide sequence. 3.The isolated polynucleotide of claim 2, wherein the first polynucleotidesequence encodes the polypeptide consisting of SEQ ID NO:2.
 4. Theisolated polynucleotide of claim 3, wherein the isolated polynucleotideconsists of the first polynucleotide sequence.
 5. The isolatedpolynucleotide of claim 2, wherein the first polynucleotide sequenceencodes the polypeptide consisting of SEQ ID NO:4.
 6. The isolatedpolynucleotide of claim 5, wherein the isolated polynucleotide consistsof the first polynucleotide sequence.
 7. The isolated polynucleotide ofclaim 2, wherein the first polynucleotide sequence encodes thepolypeptide consisting of SEQ ID NO:6.
 8. The isolated polynucleotide ofclaim 7, wherein the isolated polynucleotide consists of the firstpolynucleotide sequence.
 9. The isolated polynucleotide of claim 2,wherein the first polynucleotide sequence encodes the polypeptideconsisting of SEQ ID NO:8.
 10. The isolated polynucleotide of claim 9,wherein the isolated polynucleotide consists of the first polynucleotidesequence.
 11. An expression vector comprising the isolatedpolynucleotide of claim
 1. 12. A host cell comprising the expressionvector of claim
 11. 13. An immunogenic composition comprising theisolated polynucleotide of claim 1 and a pharmaceutically acceptablecarrier.
 14. The immunogenic composition of claim 13, further comprisingan adjuvant.
 15. An isolated polynucleotide comprising a firstpolynucleotide or the full complement of the first polynucleotidesequence, wherein the first polynucleotide sequence is selected from thegroup consisting of SEQ ID NO:1, 3, 5 or
 7. 16. The isolatedpolynucleotide of claim 15, wherein the isolated polynucleotidecomprises the first polynucleotide sequence.
 17. The isolatedpolynucleotide of claim 16, wherein the first polynucleotide sequenceconsists of SEQ ID NO:1.
 18. The isolated polynucleotide of claim 17,wherein the isolated polynucleotide consists of the first polynucleotidesequence.
 19. The isolated polynucleotide of claim 16, wherein the firstpolynucleotide sequence consists of SEQ ID NO:3.
 20. The isolatedpolynucleotide of claim 19, wherein the isolated polynucleotide consistsof the first polynucleotide sequence.
 21. The isolated polynucleotide ofclaim 16, wherein the first polynucleotide sequence consists of SEQ IDNO:5.
 22. The isolated polynucleotide of claim 21, wherein the isolatedpolynucleotide consists of the first polynucleotide sequence.
 23. Theisolated polynucleotide of claim 16, wherein the first polynucleotidesequence consists of SEQ ID NO:1.
 24. The isolated polynucleotide ofclaim 23, wherein the isolated polynucleotide consists of the firstpolynucleotide sequence.
 25. An expression vector comprising theisolated polynucleotide of claim
 15. 26. A host cell comprising theexpression vector of claim
 25. 27. An immunogenic composition comprisingthe isolated polynucleotide of claim 15 and a pharmaceuticallyacceptable carrier.
 28. The immunogenic composition of claim 27, furthercomprising an adjuvant.